Methods and compositions for stabilizing nucleic acid-nucleotide-polymerase complexes

ABSTRACT

Methods, compositions, kits and apparatuses that include a fluid, the fluid containing a ternary complex and Li + , wherein the ternary complex includes a primed template nucleic acid, a polymerase, and a nucleotide cognate for the next correct base for the primed template nucleic acid molecule. As an alternative or addition to Li + , the fluid can contain betaine or a metal ion that inhibits polymerase catalysis such as Ca 2+ . In addition to Li + , the fluid can contain polyethylenimine (PEI) with or without betaine.

CROSS REFERENCE TO RELATED APPLICATION

This application is based on, and claims the benefit of, U.S.Provisional Application No. 62/662,888, filed Apr. 26, 2018, which isincorporated herein by reference in its entirety.

BACKGROUND

The present disclosure relates generally to capture, detection andsequencing of nucleic acids. More specifically, the disclosure relatesto formation and detection of ternary complexes that each include aprimed template nucleic acid, polymerase, and cognate nucleotide, forexample, in a Sequencing By Binding™ (SBB™) method.

SBB™ technology employs transient binding of a polymerase and cognatenucleotide to a primed template nucleic acid as a means to identify thetemplate base that is at the end of the primer. Serial steps ofextending the primer and detecting the next template base allow thesequence of the template to be determined. Exemplary SBB™ techniques aredisclosed, for example, in commonly owned U.S. Pat. App. Pubs.2017/0022553 A1 and 2018/0044727 A1; and U.S. Pat. App. Ser. No.15/873,343 (published as US 2018/0208983 A1) and Ser. No. 15/851,383(published as US 2018/0187245 A1), each of which is incorporated hereinby reference.

A difficulty of SBB™ technology is that a ternary complex is anequilibrium binding product. An equilibrium binding product coexists insolution with non-bound binding partners. Removal of non-bound bindingpartners from an equilibrium reaction causes the binding product todissociate. When using labeled nucleotides in the SBB™ procedure,non-bound, labeled nucleotide provides a desired function of maintainingthe equilibrium that, in turn, maintains the ternary complex. However,the non-bound, labeled nucleotide undesirably produces background signalthat can obscure detection of the ternary complex. A similar difficultycan arise when using labeled polymerase in lieu of labeled nucleotides.

What is needed is a method to maintain detectable levels of ternarycomplexes while decreasing the concentration of labeled backgroundcomponents. The present invention satisfies this need and providesrelated advantages as well.

BRIEF SUMMARY

The present disclosure provides methods, compositions, kits andapparatuses that include a fluid, the fluid containing a ternary complexand Li⁺, wherein the ternary complex includes a primed template nucleicacid, a polymerase, and a nucleotide cognate for the next correct basefor the primed template nucleic acid molecule. As an alternative oraddition to Li⁺, the fluid can contain betaine and/or a metal ion thatinhibits polymerase catalysis such as Ca²⁺. In addition to Li⁺, thefluid can contain polyethylenimine (PEI) with or without betaine.

Also provided is a method of detecting a primed template nucleic acidthat includes the steps of (a) providing a fluid containing a ternarycomplex and Li⁺, wherein the ternary complex includes a primed templatenucleic acid, a polymerase, and a nucleotide cognate for the nextcorrect base for the primed template nucleic acid molecule; and (b)detecting the ternary complex while it is in the fluid containing theLi⁺. Optionally, the method further includes a step of (c) identifyingthe next correct base for the primed template nucleic acid molecule fromthe result of step (d). As an alternative or addition to Li⁺, the fluidcan contain betaine and/or an inhibitory metal ion such as Ca²⁺. Inaddition to Li⁺, the fluid can contain PEI with or without betaine.

A method of detecting a primed template nucleic acid can include thesteps of: (a) providing a mixture that includes a ternary complex, theternary complex including a primed template nucleic acid, a polymerase,and a nucleotide cognate for the next base of the primed templatenucleic acid, wherein the mixture further includes excess polymerase ofthe same type present in the ternary complex and excess nucleotide ofthe same type present in the ternary complex; (b) replacing the excesspolymerase and the excess nucleotide with a fluid containing Li⁺; and(c) detecting the ternary complex while it is in contact with the fluidcontaining Li⁺. Optionally, the method further includes a step of (d)identifying the next correct base for the primed template nucleic acidmolecule from the result of step (c). As an alternative or addition toLi⁺, the fluid can contain betaine and/or an inhibitory metal ion suchas Ca²⁺. In addition to Li⁺, the fluid can contain PEI with or withoutbetaine.

In some embodiments, a method of the present disclosure can include astep of extending a primer. For example, a method of detecting a primedtemplate nucleic acid can include the steps of: (a) providing a fluidcontaining a ternary complex and Li⁺, wherein the ternary complexincludes a primed template nucleic acid, a polymerase, and a nucleotidecognate for the next correct base for the primed template nucleic acidmolecule; (b) detecting the ternary complex while it is in the fluidcontaining the Li⁺, (c) identifying the next correct base for the primedtemplate nucleic acid molecule from the result of step (b); and (d)extending the primer of primed template nucleic acid. Optionally, themethod can further include a step of (e) repeating steps (a) through (d)using the primed template nucleic acid having the extended primer inplace of the primed template nucleic acid. As an alternative or additionto Li⁺, the fluid can contain betaine and/or an inhibitory metal ionsuch as Ca²⁺. In addition to Li⁺, the fluid can contain PEI with orwithout betaine.

In another example of a method that includes a primer extension step,the steps of the method can include: (a) providing a mixture thatincludes a ternary complex, the ternary complex including a primedtemplate nucleic acid, a polymerase, and a nucleotide cognate for thenext base of the primed template nucleic acid, wherein the mixturefurther includes excess polymerase of the same type present in theternary complex and excess nucleotide of the same type present in theternary complex; (b) replacing the excess polymerase and the excessnucleotide with a fluid containing Li⁺; (c) detecting the ternarycomplex while it is in contact with the fluid containing Li⁺; (d)identifying the next correct base for the primed template nucleic acidmolecule from the result of step (c); and (e) extending the primer ofprimed template nucleic acid. Optionally, the method can further includea step of (f) repeating steps (a) through (e) using the primed templatenucleic acid having the extended primer in place of the primed templatenucleic acid. As an alternative or addition to Li⁺, the fluid cancontain betaine and/or an inhibitory metal ion such as Ca²⁺. In additionto Li⁺, the fluid can contain PEI with or without betaine.

In yet another example of a method that includes a primer extensionstep, the steps of the method can include: (a) providing a mixture thatincludes a ternary complex, the ternary complex including a primedtemplate nucleic acid, a polymerase, and a nucleotide cognate for thenext base of the primed template nucleic acid, wherein the mixturefurther includes excess nucleotide of the same type present in theternary complex; (b) replacing the excess nucleotide with a fluidcontaining Li⁺; (c) detecting the ternary complex while it is in contactwith the fluid containing Li⁺; (d) identifying the next correct base forthe primed template nucleic acid molecule from the result of step (c);and (e) extending the primer of primed template nucleic acid.Optionally, the method can further include a step of (f) repeating steps(a) through (e) using the primed template nucleic acid having theextended primer in place of the primed template nucleic acid. As analternative or addition to Li⁺, the fluid can contain betaine and/or aninhibitory metal ion such as Ca²⁺. In addition to Li⁺, the fluid cancontain PEI with or without betaine.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a plot of ‘on’ and ‘off’ signal intensities for variousexamination conditions.

FIG. 1B shows a plot of purity for various examination conditions.

FIG. 2 shows a plot of ‘on’ and ‘off’ signal intensities for variousexamination conditions.

FIG. 3 shows a plot of purity for various examination conditions.

FIG. 4A shows a plot of ‘on’ and ‘off’ signal intensities for fourdifferent base types over 20 sequencing cycles carried out in theabsence of LiCl.

FIG. 4B shows a plot of ‘on’ and ‘off’ signal intensities for fourdifferent base types over 20 sequencing cycles carried out in thepresence of 5 mM LiCl.

FIG. 4C shows a plot of ‘on’ and ‘off’ signal intensities for fourdifferent base types over 20 sequencing cycles carried out in thepresence of 50 mM LiCl.

FIG. 5A shows a plot of purity for four different base types over 20sequencing cycles carried out in the absence of LiCl.

FIG. 5B shows a plot of purity for four different base types over 20sequencing cycles carried out in the presence of 5 mM LiCl.

FIG. 5C shows a plot of purity for four different base types over 20sequencing cycles carried out in the presence of 50 mM LiCl.

FIG. 6A shows a plot of signal to noise ratio over 20 sequencing cyclescarried out in the absence of LiCl.

FIG. 6B shows a plot of signal to noise ratio over 20 sequencing cyclescarried out in the presence of 5 mM LiCl.

FIG. 6C shows a plot of signal to noise ratio over 20 sequencing cyclescarried out in the presence of 50 mM LiCl.

FIG. 7A shows a plot of average error rate over 20 sequencing cyclescarried out in the absence of LiCl.

FIG. 7B shows a plot of average error rate over 20 sequencing cyclescarried out in the presence of 5 mM LiCl.

FIG. 7C shows a plot of average error rate over 20 sequencing cyclescarried out in the presence of 50 mM LiCl.

FIG. 8 shows percent loss in signal intensity for ternary complexesafter 60 seconds in the presence of varying combinations of Lithium,Calcium or betaine.

FIG. 9 shows a plot of purity values for sequencing cycles run in thepresence of varying combinations of Lithium, Calcium or betaine.

FIG. 10 shows the results of a stability test for ternary complexes inthe presence and absence of PEI.

FIG. 11 shows the results of a stability test for ternary complexes inthe presence of different concentrations of PEI.

DETAILED DESCRIPTION

Described herein is a procedure permitting rapid and efficientidentification of cognate nucleotides among members of a population ofprimed template nucleic acid molecules. As set forth below, this can beaccomplished in different ways.

Generally, preformed ternary complexes harboring a detectable label(e.g., labeled nucleotide) can be separated from non-complexedpolymerases and/or nucleotides (e.g., non-complexed labeled nucleotidesor non-complexed labeled polymerases) and then imaged in the presence ofa stabilizing fluid to make a nucleotide identification. The stabilizingfluid advantageously permits ternary complex detection over an extendedperiod. It is to be understood that there are many ways in which ternarycomplexes can be formed, and that the manner in which the ternarycomplex is formed does not generally affect usefulness of thestabilizing procedure set forth herein.

Embodiments of the methods set forth herein can be used to form areversible complex (e.g. a ternary complex) by contacting a polymerase,nucleotide and primed template nucleic acid under conditions that allowthe three components to form a complex while precluding extension at the3′-end of the primer. Extension can be precluded by a number of meansset forth herein including, but not limited to, presence of a terminatormoiety on the primer, presence of a polymerase inhibitor such as aninhibitory metal ion, absence of a catalytic metal ion and/or use of apolymerase variant that is inhibited from primer extension activity(e.g. due to a mutation in the catalytic domain of the polymerase). Theprimed template nucleic acid can be immobilized to a solid support ifdesired. The extent of ternary complex formation reflects an equilibriumbinding condition resulting from the presence of the different bindingcomponents (i.e., polymerase and cognate nucleotide) at their associatedconcentrations. Although the net effect of equilibrium is that complexesthat form appear to be stable during this binding step, individualcomplexes actually are in a state of flux. Indeed, components of thecomplex can be in a situation where they are continuously associatingand dissociating with the blocked primed template nucleic acid moleculeat equilibrium but there is no net change in concentration of freecomponents and bound components.

Optionally, ternary complexes once formed can be contacted with astabilizing fluid prior to detection. The stabilizing fluid can be usedto change the chemical environment containing the ternary complexes.This means that the ternary complexes formed under one condition can bedetected under a different condition. Although not necessarily wishingto be limited by the proposed mechanism, the stabilizing fluid can slowthe dissociation of ternary complexes that otherwise occurs in theabsence of one or more binding components (e.g., nucleotide and/orpolymerase), thereby allowing detection of the ternary complexes over anextended timeframe. A stabilizing fluid can have other effects thatimprove the ability to detect or manipulate ternary complexes. Forexample, some stabilizing fluids can inhibit formation of binarycomplexes between polymerase and primed template nucleic acid (i.e.absent a cognate nucleotide) and/or otherwise act to increase the ratioof ternary complex to binary complex.

As disclosed herein, ternary complexes including a primed templatenucleic acid, cognate nucleotide (optionally including a detectablelabel), and a polymerase (optionally including a detectable label) canbe detected during or after a wash step that separates the ternarycomplex from non-complexed polymerase and non-complexed nucleotides. Thewash step can be an imaging wash step employing an aqueous stabilizingfluid that includes a stabilizing agent that maintains the complexes(e.g., relative to the fluid lacking the stabilizing agent). Exemplarystabilizing agents include Lithium (Li⁺), betaine, and/or an inhibitorymetal ion such as Ca²⁺. A particularly useful stabilizing agent includesLi⁺ in combination with polyethylenimine (PEI) and optionally furthercombined with betaine. It will be understood that Li⁺, betaine, andother metal ions (e.g. Ca²⁺) need not function as a stabilizing agent tobe useful in a method or composition set forth herein. Accordingly,stabilization of ternary complexes is an optional use for these andother reagents set forth herein.

The presence of a stabilizing agent can permit removal of excess labeledcomponents (e.g., labeled nucleotides or labeled polymerase) from areaction vessel, while maintaining ternary complexes in the vessel fordetection or other uses. Accordingly, ternary complexes immobilizedwithin a vessel, such as a flow cell, can be washed with a stabilizingfluid and detected while in contact with the stabilizing fluid. Anadvantage of using the stabilizing fluid wash is that the ternarycomplex can be detected substantially in the absence of excess,non-complexed binding components (e.g. labeled nucleotides and/orlabeled polymerase) that would undesirably increase background signals.Thus, detecting ternary complexes during a stabilizing wash step caninvolve detecting the complexes under changed conditions (e.g.,conditions different from the equilibrium that resulted from formationof the ternary complexes). During the wash step the net forward reactionthat previously lead to ternary complex formation no longer occurs or isat least substantially slowed.

Washing ternary complexes by flowing a nucleotide-free andpolymerase-free stabilizing fluid through a flow cell can removenon-complexed labeled nucleotide and polymerase and reduce non-specificbackground signals (e.g., fluorescent background) while preservingdetectability of pre-formed or existing ternary complexes. For example,ternary complexes contained within a flow cell can be detected after aperiod of contact with the stabilizing fluid of at least 30 seconds, 1minute, 5 minutes, 10 minutes or longer. Alternatively or additionally,it may be desirable to detect the stabilized ternary complexes after aperiod of contact with the stabilizing fluid of at most 10 minutes, 5minutes, 1 minute, 30 seconds or less.

The compositions and methods used in several embodiments set forthherein exploit the binding specificity of a ternary complex thatincludes a polymerase, a primed template nucleic acid, and a cognatenucleotide. This specificity can be used to identify the next correctnucleotide for the primed template nucleic acid by identifying thenucleotide present in the ternary complex. By this approach, blockingthe primer from extension at its 3′-end, and then detecting formation ofa ternary complex while precluding phosphodiester bond formation,optionally under equilibrium binding conditions, can occur in the samereaction mixture and without intervening reagent exchange or wash steps.Alternatively, formation of a ternary complex can be detected during animaging wash step when excess nucleotides and polymerase have beenremoved from the system. The optional presence of catalytic metal ionsduring formation and examination of ternary complexes can mimic a morenatural ternary complex condition, and so provides an added benefit overmethods that omit or replace catalytic ions. The aggregated result isincreased speed of single nucleotide identification (e.g. in agenotyping procedure or single sequencing cycle) and sequenceidentification (e.g. in a cyclic process using repeated cycles ofcognate nucleotide identification and primer extension).

The present disclosure exemplifies and describes several aspects ofternary complex stabilization in the context of a Sequencing By Binding™technique. It will be understood that the compositions and methods setforth herein need not be limited to nucleic acid sequencing. Forexample, this disclosure provides methods for interrogating a singlenucleotide site in a primed template nucleic acid. Interrogation of asingle nucleotide site can be useful for detecting a variant at a singlesite (e.g., a single nucleotide polymorphism or SNP), for example, in agenotyping method. Typically, a genotyping method is carried out using atemplate nucleic acid with a known genetic locus, but for which anallelic variation at the locus is to be determined. Alternatively,identification of a single nucleotide site can be useful for evaluatingcharacteristics of a target polymerase, such as specificity of thepolymerase for binding to a correct nucleotide. Methods that interrogateonly a single nucleotide site in a template nucleic acid can be carriedout using a single cycle of a Sequencing By Binding™ method set forthherein. Optionally, a single nucleotide site can be interrogated usingmethods or reagents of the present disclosure in combination withmethods or reagents set forth in commonly owned U.S. Pat. No. 9,932,631and U.S. provisional application having Ser. No. 62/448,630, each ofwhich is incorporated herein by reference.

Another exemplary application of the compositions and methods set forthherein is polymerase-based capture of allelic variants. The capturemethods exploit the specificity with which a polymerase can form astabilized ternary complex with a primed template and a next correctnucleotide. For example, a stabilized ternary complex can be formedbetween a polymerase, target allele and cognate nucleotide for theallele. Polymerase specificity allows a target allele to be separatedfrom other nucleic acids, including for example, other alleles thatdiffer from the target allele by a single nucleotide. For example, aternary complex can be formed between a polymerase, a primed templateencoding a target single nucleotide polymorphism (SNP) allele and acognate nucleotide for the SNP allele. Capture of the ternary complexwill result in selective capture of the SNP allele, compared to anon-target SNP allele at the same locus, because the cognate nucleotideis selective for the target SNP when forming a ternary complex with thepolymerase. Use of a stabilizing agent can be used to improve thesecapture methods and methods set forth in U.S. patent application Ser.No. 15/701,358, now published as US Pat. App. Pub. No. 2018/0208922 A1,which is incorporated herein by reference.

Terms used herein will be understood to take on their ordinary meaningin the relevant art unless specified otherwise. Several terms usedherein, and their meanings, are set forth below.

As used herein, the term “array” refers to a population of moleculesthat are attached to one or more solid supports such that the moleculesat one feature can be distinguished from molecules at other features. Anarray can include different molecules that are each located at differentaddressable features on a solid support. Alternatively, an array caninclude separate solid supports each functioning as a feature that bearsa different molecule, wherein the different molecules can be identifiedaccording to the locations of the solid supports on a surface to whichthe solid supports are attached, or according to the locations of thesolid supports in a liquid such as a fluid stream. The molecules of thearray can be, for example, nucleotides, nucleic acid primers, nucleicacid templates, primed nucleic acid templates or nucleic acid enzymessuch as polymerases, ligases, exonucleases or combinations thereof.

As used herein, the term “betaine” means a zwitterionic molecule havingcharge-separated forms with an onium atom which bears no hydrogen atomsand that is not adjacent to the anionic atom. The anionic atom can be acarboxylate group. An ammonium betaine has a cationic functional groupthat includes a quaternary ammonium. A particularly useful ammoniumbetaine is N,N,N-trimethylglycine (TMG). A phosphonium betaine has acationic functional group that includes a phosphonium cation.

As used herein, the term “binary complex” refers to an intermolecularassociation between a polymerase and a primed template nucleic acid,exclusive of a nucleotide molecule such as a next correct nucleotide ofthe primed template nucleic acid.

As used herein, the term “blocking moiety,” when used in reference to anucleotide, means a part of the nucleotide that inhibits or prevents the3′ oxygen of the nucleotide from forming a covalent linkage to a nextcorrect nucleotide during a nucleic acid polymerization reaction. Theblocking moiety of a “reversible terminator” nucleotide can be removedfrom the nucleotide analog, or otherwise modified, to allow the3′-oxygen of the nucleotide to covalently link to a next correctnucleotide. This process is referred to as “deblocking” the nucleotideanalog. Such a blocking moiety is referred to herein as a “reversibleterminator moiety.” Exemplary reversible terminator moieties are setforth in U.S. Pat. Nos. 7,427,673; 7,414,116; 7,057,026; 7,544,794 or8,034,923; or PCT publications WO 91/06678 or WO 07/123744, each ofwhich is incorporated herein by reference. A nucleotide that has ablocking moiety or reversible terminator moiety can be at the 3′ end ofa nucleic acid, such as a primer, or can be a monomer that is notcovalently attached to a nucleic acid.

As used herein, the term “catalytic metal ion” refers to a metal ionthat facilitates phosphodiester bond formation between the 3′-OH of anucleic acid (e.g., a primer) and the phosphate of an incomingnucleotide by a polymerase. A “divalent catalytic metal cation” is acatalytic metal ion having a valence of two. Catalytic metal ions can bepresent at concentrations that stabilize formation of a complex betweena polymerase, nucleotide, and primed template nucleic acid, referred toas non-catalytic concentrations of a metal ion insofar as phosphodiesterbond formation does not occur. Catalytic concentrations of a metal ionrefer to the amount of a metal ion sufficient for polymerases tocatalyze the reaction between the 3′-OH group of a nucleic acid (e.g., aprimer) and the phosphate group of an incoming nucleotide.

The term “comprising” is intended herein to be open-ended, including notonly the recited elements, but further encompassing any additionalelements.

As used herein, the terms “cycle” or “round,” when used in reference toa sequencing procedure, refer to the portion of a sequencing run that isrepeated to indicate the presence of a nucleotide. Typically, a cycle orround includes several steps such as steps for delivery of reagents,washing away unreacted reagents and detection of signals indicative ofchanges occurring in response to added reagents.

As used herein, the term “diffusional exchange,” when used in referenceto members of a binding complex, refers to the ability of the members tomove in a fluid to associate with, or dissociate from, each other.Diffusional exchange can occur when there are no barriers that preventthe members from interacting with each other to form a complex. However,diffusional exchange is understood to exist even if diffusion isretarded, reduced or altered so long as access is not absolutelyprevented.

As used herein, the term “each,” when used in reference to a collectionof items, is intended to identify an individual item in the collectionbut does not necessarily refer to every item in the collection.Exceptions can occur if explicit disclosure or context clearly dictatesotherwise.

As used herein, “equilibrium” refers to a state of balance due to theequal action of opposing forces. For example, a ternary complex formedbetween a primed template nucleic acid, polymerase, and cognatenucleotide is in equilibrium with non-bound polymerase and cognatenucleotide when the rate of formation of the ternary complex is balancedby the rate of its dissolution. Under this condition, the reversiblebinding reaction ceases to change its net ratio of products toreactants. If the rate of a forward reaction (e.g., ternary complexformation) is balanced by the rate of a reverse reaction (e.g., ternarycomplex dissociation), then there is no net change.

As used herein, the term “excess,” when used in reference to componentsthat are capable of forming a complex in a binding reaction, refers tocomponents that are not in a bound state. Taking as an example, areaction that forms a ternary complex, polymerases or nucleotides thatare free in solution in the reaction vessel with the ternary complex areexcess polymerases and nucleotides.

As used herein, the term “exogenous,” when used in reference to a moietyof a molecule, means a chemical moiety that is not present in a naturalanalog of the molecule. For example, an exogenous label of a nucleotideis a label that is not present on a naturally occurring nucleotide.Similarly, an exogenous label that is present on a polymerase is notfound on the polymerase in its native milieu.

As used herein, the term “extension,” when used in reference to anucleic acid, means a process of adding at least one nucleotide to the3′ end of the nucleic acid. The term “polymerase extension,” when usedin reference to a nucleic acid, refers to a polymerase catalyzed processof adding at least one nucleotide to the 3′ end of the nucleic acid. Anucleotide or oligonucleotide that is added to a nucleic acid byextension is said to be incorporated into the nucleic acid. Accordingly,the term “incorporating” can be used to refer to the process of joininga nucleotide or oligonucleotide to the 3′ end of a nucleic acid byformation of a phosphodiester bond.

As used herein, the term “extendable,” when used in reference to anucleotide, means that the nucleotide has an oxygen or hydroxyl moietyat the 3′ position, and is capable of forming a covalent linkage to anext correct nucleotide if and when incorporated into a nucleic acid. Anextendable nucleotide can be at the 3′ position of a primer or it can bea monomeric nucleotide. A nucleotide that is extendable will lackblocking moieties such as reversible terminator moieties.

As used herein, the term “feature,” when used in reference to an array,means a location in an array where a particular molecule is present. Afeature can contain only a single molecule or it can contain apopulation of several molecules of the same species (i.e. an ensemble ofthe molecules). Alternatively, a feature can include a population ofmolecules that are different species (e.g. a population of ternarycomplexes having different template sequences). Features of an array aretypically discrete. The discrete features can be contiguous or they canhave spaces between each other. An array useful herein can have, forexample, features that are separated by less than 100 microns, 50microns, 10 microns, 5 microns, 1 micron, or 0.5 micron. Alternativelyor additionally, an array can have features that are separated bygreater than 0.5 micron, 1 micron, 5 microns, 10 microns, 50 microns or100 microns. The features can each have an area of less than 1 squaremillimeter, 500 square microns, 100 square microns, 25 square microns, 1square micron or less.

As used herein, the term “label” refers to a molecule or moiety thereofthat provides a detectable characteristic. The detectable characteristiccan be, for example, an optical signal such as absorbance of radiation,luminescence or fluorescence emission, luminescence or fluorescencelifetime, luminescence or fluorescence polarization, or the like;Rayleigh and/or Mie scattering; binding affinity for a ligand orreceptor; magnetic properties; electrical properties; charge; mass;radioactivity or the like. Exemplary labels include, without limitation,a fluorophore, luminophore, chromophore, nanoparticle (e.g., gold,silver, carbon nanotubes), heavy atoms, radioactive isotope, mass label,charge label, spin label, receptor, ligand, or the like.

As used herein, the term “next correct nucleotide” refers to thenucleotide type that will bind and/or incorporate at the 3′ end of aprimer to complement a base in a template strand to which the primer ishybridized. The base in the template strand is referred to as the “nextbase” and is immediately 5′ of the base in the template that ishybridized to the 3′ end of the primer. The next correct nucleotide canbe referred to as the “cognate” of the next base and vice versa. Cognatenucleotides that interact specifically with each other in a ternarycomplex or in a double stranded nucleic acid are said to “pair” witheach other. A nucleotide having a base that is not complementary to thenext template base is referred to as an “incorrect”, “mismatch” or“non-cognate” nucleotide.

As used herein, the term “inhibitory metal ion” refers to a metal ionthat, when in the presence of a polymerase enzyme, inhibitsphosphodiester bond formation needed for chemical incorporation of anucleotide into a primer. An inhibitory metal ion may interact with apolymerase, for example, via competitive binding compared to catalyticmetal ions. A “divalent inhibitory metal ion” is an inhibitory metal ionhaving a valence of two. Examples of divalent inhibitory metal ionsinclude, but are not limited to, Ca²⁺, Zn²⁺, Co²⁺, Ni²⁺, and Sr²⁺. Thetrivalent Eu³⁺ and Tb³⁺ ions are inhibitory metal ions having a valenceof three.

As used herein, the term “nucleotide” can be used to refer to a nativenucleotide or analog thereof. Examples include, but are not limited to,nucleotide triphosphates (NTPs) such as ribonucleotide triphosphates(rNTPs), deoxyribonucleotide triphosphates (dNTPs), or non-naturalanalogs thereof such as dideoxyribonucleotide triphosphates (ddNTPs) orreversibly terminated nucleotide triphosphates (rtNTPs).

As used herein, the term “polyethylenimine” or “PEI” refers to a polymerwith repeating unit composed [NCH₂CH₂]_(n). Linear polyethyleneiminescontain all secondary amines (i.e. [NHCH₂CH₂]_(n)), in contrast tobranched PEIs which contain primary, secondary and/or tertiary aminogroups. The polymer can be in a polycationic form. Polyethylenimine isalso known in the art as poly(iminoethylene), polyaziridine, orpoly[imino(1,2-ethanediyl)].

As used herein, the term “polymerase” can be used to refer to a nucleicacid synthesizing enzyme, including but not limited to, DNA polymerase,RNA polymerase, reverse transcriptase, primase and transferase.Typically, the polymerase has one or more active sites at whichnucleotide binding and/or catalysis of nucleotide polymerization mayoccur. The polymerase may catalyze the polymerization of nucleotides tothe 3′ end of the first strand of the double stranded nucleic acidmolecule. For example, a polymerase catalyzes the addition of a nextcorrect nucleotide to the 3′ oxygen group of the first strand of thedouble stranded nucleic acid molecule via a phosphodiester bond, therebycovalently incorporating the nucleotide to the first strand of thedouble stranded nucleic acid molecule. Optionally, a polymerase need notbe capable of nucleotide incorporation under one or more conditions usedin a method set forth herein. For example, a mutant polymerase may becapable of forming a ternary complex but incapable of catalyzingnucleotide incorporation.

As used herein, the term “primed template nucleic acid” refers to anucleic acid hybrid having a double stranded region such that one of thestrands has a 3′-end that can be extended by a polymerase. The twostrands can be parts of a contiguous nucleic acid molecule (e.g. ahairpin structure) or the two strands can be separable molecules thatare not covalently attached to each other.

As used herein, the term “primer” refers to a nucleic acid having asequence that binds to a nucleic acid at or near a template sequence.Generally, the primer binds in a configuration that allows replicationof the template, for example, via polymerase extension of the primer.The primer can be a first portion of a nucleic acid molecule that bindsto a second portion of the nucleic acid molecule, the first portionbeing a primer sequence and the second portion being a primer bindingsequence (e.g. a hairpin primer). Alternatively, the primer can be afirst nucleic acid molecule that binds to a second nucleic acid moleculehaving the template sequence (e.g. a dissociable primer). A primer canconsist of DNA, RNA or analogs thereof.

As used herein, a “reaction vessel” is a container that isolates onereagent or reaction (e.g., a binding reaction; an incorporationreaction; etc.) from another, or that provides a space in which areaction can take place. Non-limiting examples of reaction vesselsuseful in connection with the disclosed technique include: flow cells,wells of a multiwell plate; microscope slides; open tubes (e.g.,capillary tubes); closed tubes (e.g. microcentrifuge tubes, test tubesor Eppendorf Tubes™); etc. Features to be monitored during bindingand/or incorporation reactions can be contained within the reactionvessel.

As used herein, the term “solid support” refers to a rigid substratethat is insoluble in aqueous liquid. The substrate can be non-porous orporous. The substrate can optionally be capable of taking up a liquid(e.g. due to porosity) but will typically be sufficiently rigid that thesubstrate does not swell substantially when taking up the liquid anddoes not contract substantially when the liquid is removed by drying. Anonporous solid support is generally impermeable to liquids or gases.Exemplary solid supports include, but are not limited to, glass andmodified or functionalized glass, plastics (including acrylics,polystyrene and copolymers of styrene and other materials,polypropylene, polyethylene, polybutylene, polyurethanes, Teflon™,cyclic olefins, polyimides etc.), nylon, ceramics, resins, Zeonor®,silica or silica-based materials including silicon and modified silicon,carbon, metals, inorganic glasses, optical fiber bundles, and polymers.

As used herein, the term “ternary complex” refers to an intermolecularassociation between a polymerase, a double stranded nucleic acid and anucleotide. Typically, the polymerase facilitates interaction between anext correct nucleotide and a template strand of the primed nucleicacid. A next correct nucleotide can interact with the template strandvia Watson-Crick hydrogen bonding. The term “stabilized ternary complex”means a ternary complex having promoted or prolonged existence or aternary complex for which disruption has been inhibited. Generally,stabilization of the ternary complex prevents covalent incorporation ofthe nucleotide component of the ternary complex into the primed nucleicacid component of the ternary complex.

As used herein, the term “type” or “species” is used to identifymolecules that share the same chemical structure. For example, a mixtureof nucleotides can include several dCTP molecules. The dCTP moleculeswill be understood to be the same type (or species) of nucleotide aseach other, but a different type (or species) of nucleotide compared todATP, dGTP, dTTP etc. Similarly, individual DNA molecules that have thesame sequence of nucleotides are the same type (or species) of DNA,whereas DNA molecules with different sequences are different types (orspecies) of DNA. The term “type” or “species” can also identify moietiesthat share the same chemical structure. For example, the cytosine basesin a template nucleic acid will be understood to have the same type (orspecies) of base as each other independent of their position in thetemplate sequence.

The embodiments set forth below and recited in the claims can beunderstood in view of the above definitions.

The present disclosure provides a method of detecting a primed templatenucleic acid. The method can include the steps of: (a) providing amixture that includes a ternary complex, the ternary complex including aprimed template nucleic acid, a polymerase, and a nucleotide cognate forthe next base of the primed template nucleic acid, wherein the mixturefurther includes excess polymerase of the same type present in theternary complex and excess nucleotide of the same type present in theternary complex; (b) replacing the excess polymerase and the excessnucleotide with a fluid containing Li⁺ and (c) detecting the ternarycomplex while it is in contact with the fluid containing Li⁺.Optionally, the method further includes a step of (d) identifying thenext correct base for the primed template nucleic acid molecule from theresult of step (c). As an alternative or addition to Li⁺, the fluid cancontain betaine and/or an inhibitory metal ion such as Ca²⁺. In additionto Li⁺, the fluid can contain polyethylenimine (PEI) with or withoutbetaine.

High concentrations of detectably labeled components can be used todrive formation of transient or reversible ternary complexes that are tobe detected. Unfortunately, non-complexed reagents harboring detectablelabels and remaining in the presence of the specific complexes cangenerate signals that confound or mask the desired detection. This isespecially problematic when the signal generated by the detectable labelis substantially similar irrespective of whether the labeled component(e.g., polymerase or nucleotide) is free in solution or included in acomplex (e.g., a ternary complex).

In particular embodiments, nucleotide concentrations substantiallyexceed polymerase concentrations in binding reaction mixtures and, assuch, procedures employing labeled nucleotide for detection of ternarycomplexes can be particularly susceptible to high backgrounds thatobscure ternary complex detection. Moreover, the dynamic nature of theternary complex (e.g., where ternary complexes are in a state of flux,forming and dissociating, and exchanging with components in theirchemical environments) can complicate examination of the ternary complexproduct when conventional aqueous wash steps are performed to removenon-complexed reagents from the system. This is because the reversiblecomplex that is to be detected can be unstable over a time period thatis used to examine or monitor the ternary complex. Two technical issuesimpact detection of multicomponent complexes when using components thatinclude detectable labels. First, signals originating from the labelednon-bound components can undesirably obscure detection of specificcomplexes. Second, conventional washing to remove one or more componentsfrom the system can promote dissociation of the reversible complexesthat are to be detected. Each of these can be a liability when gatheringsequencing data.

The importance of maintaining stability of a bound complex whiledetecting the complex can be appreciated in the context of array-basedapplications, where multiple images are acquired along the surface ofthe array. For example, a flow cell can include an array having asurface area greater than a single field of view for an optical imagingsystem. As a consequence, an optical system may acquire images ofdifferent parts of the array by a scanning or stepping process. Iftransient complexes to be monitored are unstable, then it is possiblethat lower quality data will be acquired for the later images comparedto earlier images. As set forth herein, this problem can be overcome bystabilizing complexes under a condition that permits acquisition of datawith high signal-to-background ratios.

A particularly useful agent for use in a method or composition of thepresent disclosure, for example, for stabilizing a ternary complex islithium. Like the other alkali metals, lithium has a single valenceelectron that is easily given up to form a cation (Li⁺). Lithium can besupplied to a reaction in salt form, for example, in the form of LiCl.Lithium, when in contact with ternary complex, can be at a concentrationof at least 5 mM, 10 mM, 25 mM, 50 mM, 100 mM, 250 mM or higher.Alternatively or additionally, lithium can be present at a concentrationof at most 250 mM, 100 mM, 50 mM, 25 mM, 10 mM, 5 mM or less.

Another useful agent for use in a method or composition of the presentdisclosure, for example, for stabilizing a ternary complex is betaine.Betaine, when in contact with ternary complex, can be at a concentrationof at least 1 mM, 10 mM, 50 mM, 100 mM, 500 mM, 1 M, 2 M, 3 M, 3.5M orhigher. Alternatively or additionally, betaine can be present at aconcentration of at most 3.5 M, 2 M, 1 M, 500 mM, 100 mM, 50 mM, 10 mM,1 mM, or less. Betaine can be used in combination with Li⁺ or in theabsence of Li⁺ to produce a stabilizing effect.

Polyethylenimine (PEI) can be used in a method or composition of thepresent disclosure, for example, for stabilizing a ternary complex. PEI,when in contact with a ternary complex, can be present at aconcentration of at least 0.0001%, 0.001%, 0.01%, 0.05%, 0.1%, 1% or 5%(w/v). Alternatively or additionally, PEI can be present at aconcentration of at most 5%, 1%, 0.1%, 0.05%, 0.01%, 0.001% or 0.0001%(w/v). PEI can be used in combination with Li⁺ to produce a stabilizingeffect on a ternary complex. In cases where PEI and Li⁺ are usedtogether, betaine can optionally be present as well, or betaine can beabsent.

Inhibitory metal ions can also be used in a method or composition of thepresent disclosure, for example, as a stabilizing agent. A particularlyuseful inhibitory metal ion is Ca²⁺. Inhibitory metal ions, when incontact with ternary complex, can be at a concentration of at least 0.1mM, 0.5 mM, 1 mM, 5 mM, 10 mM, 25 mM, 50 mM, 100 mM, or higher.Alternatively or additionally, inhibitory metal ions can be present at aconcentration of at most 100 mM, 50 mM, 25 mM, 10 mM, 5 mM, 1 mM, 0.5mM, 0.1 mM or less. Ca²⁺ can be used alone or in combination with one ormore of Li⁺, betaine and PEI.

A solution that is used for stabilizing a ternary complex, for example,a solution that contains Lithium, betaine, PEI and/or an inhibitorymetal ion, can be buffered at a desired pH. For example, the pH of thesolution can be at least 7.0, 7.5, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6,8.7, 8.8, 8.9 or 9.0. Alternatively or additionally, the buffer can beselected to maintain the pH to be at most 9.0, 8.9, 8.8, 8.7, 8.6, 8.5,8.4, 8.3, 8.2, 8.1, 8.0, 7.5, or 7.0. Useful buffers include those setforth in the Examples below or those having an appropriate pKa for thedesired pH range, including for example, MOPS (pKa 7.2), BES (pKa 7.2),TES (pKa 7.5), Triethanolamine (pKa 7.8), EPPS or HEPPS (pKA 8.0), TRIS(pKa 8.1), Tricene (pKa 8.1), Glycylglycine (pKa 8.3), Bicine (pKa 8.3),TAPPS (pKa 8.4), Morpholine (pKa 8.5), N-Methyldiethanolamine (pKa 8.5),2-amino-2-methyl-1,3-propanediol (pKa 8.8), Diethanolamine (pKa 8.9), orAMPSO (pKa 9.1). A buffer can be present at an effective concentrationfor stabilizing ternary complexes, for example, between about 25 mM and250 mM, between about 25 mM and 100 mM, between about 40 mM and 80 mM orother ranges.

A solution that is used for stabilizing a ternary complex, for example,a solution that contains Lithium, betaine, PEI and/or an inhibitorymetal ion, can include a salt such as those set forth in the Examplessection below. Particularly useful salts include, but are not limited toNaCl, KCl, K-acetate, NH₄-acetate, K-glutamate, NH₄Cl, or (NH₄HSO₄). Thesalts can be present at an effective concentration for stabilizingternary complexes including, for example, at least 10 mM, 25 mM, 50 mM,100 mM, 250 mM or higher. Alternatively or additionally, the saltconcentration can be at most 250 mM, 100 mM, 50 mM, 25 mM or 10 mM.

A solution that is used for stabilizing a ternary complex, for example,a solution that contains Lithium, betaine, PEI and/or an inhibitorymetal ion, can include other components such as those that providedesired viscosity or molecular crowding. Exemplary components includefor example, polysaccharides such as sucrose, Ficoll, or dextran;proteins such as lysozyme, albumin or casein; or polymers such aspolyvinyl alcohol, polyethyleneglycols (PEG 2050, PEG 4600, PEG 6000,PEG 8000, PEG 20000). A viscous or molecular crowding agent can bepresent at a concentration of at least about 0.5%, 1%, 3%, 5%, 10% ormore. Alternatively or additionally, the concentration can be at most10%, 5%, 3%, 2%, 1% or 0.5%.

Other useful components to include in a solution for stabilizing ternarycomplexes include antifade or photoprotective reagents such as DABCO(1,4-diazabicyclo[2.2.2]octane), ascorbate, gallic acid or derivativesthereof. Other useful antifade and photoprotective reagents include, forexample, those set forth in U.S. Pat. Nos. 7,993,895; 9,115,353;10,036,011, each of which is incorporated herein by reference. Suchreagents are particularly useful when present in solutions that aredetected via optical methods such as luminescence and fluorescence. Amethod of this disclosure can include one or more steps for forming anddetecting a ternary complex. Embodiments of the methods exploit thespecificity with which a polymerase can form a stabilized ternarycomplex with a primed template nucleic acid and a next correctnucleotide. The next correct nucleotide can be non-covalently bound tothe stabilized ternary complex, interacting with the other members ofthe complex solely via non-covalent interactions. Useful methods andcompositions for forming a stabilized ternary complex are set forth infurther detail below and in commonly owned U.S. Pat. App. Pub. Nos.2017/0022553 A1 or 2018/0044727 A1; US Pat. App. Pub. No. 2018/0187245A1, which claims priority to U.S. Pat. App. Ser. Nos. 62/440,624 or USPat. App. Pub. No. 2018/0208983 A1, which claims priority to 62/450,397,each of which is incorporated herein by reference. Typically, formationand detection of ternary complex is separated from a step of extendingthe primer, for example, due to reagent exchange between the steps.However, in some embodiments the binding, detection and extension stepscan occur in the same mixture.

While a ternary complex can form between a polymerase, primed templatenucleic acid and next correct nucleotide in the absence of certaincatalytic metal ions (e.g., Mg²⁺), chemical addition of the nucleotideis inhibited in the absence of the catalytic metal ions. Low ordeficient levels of catalytic metal ions, causes non-covalentsequestration of the next correct nucleotide in a stabilized ternarycomplex. Other methods disclosed herein also can be used to produce astabilized ternary complex.

Optionally, a stabilized ternary complex can be formed when the primerof the primed template nucleic acid includes a blocking moiety (e.g. areversible terminator moiety) that precludes enzymatic incorporation ofan incoming nucleotide into the primer. The interaction can take placein the presence of stabilizers, whereby the polymerase-nucleic acidinteraction is stabilized in the presence of the next correctnucleotide. The primer of the primed template nucleic acid optionallycan be either an extendible primer, or a primer blocked from extensionat its 3′-end (e.g., blocking can be achieved by the presence of areversible terminator moiety on the 3′-end of the primer). The primedtemplate nucleic acid, the polymerase and the cognate nucleotide arecapable of forming a stabilized ternary complex when the base of thecognate nucleotide is complementary to the next base of the primedtemplate nucleic acid.

As set forth above, conditions that favor or stabilize a ternary complexcan be provided by the presence of a blocking group that precludesenzymatic incorporation of an incoming nucleotide into the primer (e.g.a reversible terminator moiety on the 3′ nucleotide of the primer) orthe absence of a catalytic metal ion. Other useful conditions includethe presence of a ternary complex stabilizing agent such as aninhibitory metal ion (e.g., a divalent or trivalent inhibitory metalion) that inhibits polymerase catalyzed nucleotide incorporation orpolymerization. Inhibitory metal ions include, but are not limited to,calcium, strontium, scandium, titanium, vanadium, chromium, iron,cobalt, nickel, copper, zinc, gallium, germanium, arsenic, selenium,rhodium, europium, and terbium ions. Optionally, conditions thatdisfavor or destabilize binary complexes (i.e. complexes betweenpolymerase and primed nucleic acid but lacking cognate nucleotide) areprovided by the presence of one or more monovalent cations and/orglutamate anions. As a further example of a stabilizing condition, apolymerase engineered to have reduced catalytic activity or reducedpropensity for binary complex formation can be used. Exemplaryengineered polymerases are set forth in US Pat. App. Pub. Nos.2017/0314072 A1 or 2018/0155698 A1, each of which is incorporated hereinby reference.

Ternary complex stabilization conditions can accentuate the differencein affinity of polymerase toward primed template nucleic acids in thepresence of different nucleotides, for example, by destabilizing binarycomplexes. Optionally, the conditions cause differential affinity of thepolymerase for the primed template nucleic acid in the presence ofdifferent nucleotides. By way of example, the conditions include, butare not limited to, high salt and glutamate ions. For example, the saltmay dissolve in aqueous solution to yield a monovalent cation, such as amonovalent metal cation (e.g., sodium ion or potassium ion). Optionally,the salt that provides the monovalent cations (e.g., monovalent metalcations) further provides glutamate ions. Optionally, the source ofglutamate ions can be potassium glutamate. In some instances, theconcentrations of potassium glutamate that can be used to alterpolymerase affinity of the primed template nucleic acid extend from 10mM to 1.6 M of potassium glutamate, or any amount in between 10 mM and1.6 M. As indicated above, high salt refers to a concentration of saltfrom 50 mM to 1.5 M salt.

It will be understood that options set forth herein for stabilizing aternary complex need not be mutually exclusive and instead can be usedin various combinations. For example, a ternary complex can bestabilized by one or a combination of means including, but not limitedto, presence of crosslinking of the polymerase domains; crosslinking ofthe polymerase to the nucleic acid; polymerase mutations that stabilizethe ternary complex; allosteric inhibition by small molecules; presenceof Li⁺, PEI, betaine, uncompetitive inhibitors, competitive inhibitors,or non-competitive inhibitors; absence of catalytic metal ions; presenceof a blocking moiety on the primer; or other means set forth herein.

Nucleic acids that are used in a method or composition herein can be DNAsuch as genomic DNA, synthetic DNA, amplified DNA, copy DNA (cDNA) orthe like. RNA can also be used such as mRNA, ribosomal RNA, tRNA or thelike. Nucleic acid analogs can also be used as templates herein. Thus,template nucleic acids used herein can be derived from a biologicalsource, synthetic source or amplification product. Primers used hereincan be DNA, RNA or analogs thereof.

Particularly useful nucleic acid templates are genome fragments thatinclude sequences identical to a portion of a genome. A population ofgenome fragments can include at least 5%, 10%, 20%, 30%, or 40%, 50%,60%, 70%, 75%, 80%, 85%, 90%, 95% or 99% of a genome. A genome fragmentcan have, for example, a sequence that is substantially identical to atleast about 25, 50, 70, 100, 200, 300, 400, 500, 600, 700, 800, 900 or1000 or more nucleotides of a genome. Alternatively or additionally, agenome fragment can have a sequence that is substantially identical tono more than 1×10⁵, 1×10⁴, 1×10³, 800, 600, 400, 200, 100, 75, 50 or 25nucleotides of a genome. A genome fragment can be DNA, RNA, or an analogthereof.

Exemplary organisms from which nucleic acids can be derived include, forexample, those from a mammal such as a rodent, mouse, rat, rabbit,guinea pig, ungulate, horse, sheep, pig, goat, cow, cat, dog, primate,human or non-human primate; a plant such as Arabidopsis thaliana, corn,sorghum, oat, wheat, rice, canola, or soybean; an algae such asChlamydomonas reinhardtii; a nematode such as Caenorhabditis elegans; aninsect such as Drosophila melanogaster, mosquito, fruit fly, honey beeor spider; a fish such as zebrafish; a reptile; an amphibian such as afrog or Xenopus laevis; a dictyostelium discoideum; a fungi such aspneumocystis carinii, Takifugu rubripes, yeast, Saccharamoycescerevisiae or Schizosaccharomyces pombe; or a plasmodium falciparum.Nucleic acids can also be derived from a prokaryote such as a bacterium,Escherichia coli, staphylococci or mycoplasma pneumoniae; an archae; avirus such as Hepatitis C virus or human immunodeficiency virus; or aviroid. Nucleic acids can be derived from a homogeneous culture orpopulation of the above organisms or alternatively from a collection ofseveral different organisms, for example, in a community or ecosystem.Nucleic acids can be isolated using methods known in the art including,for example, those described in Sambrook et al., Molecular Cloning: ALaboratory Manual, 3rd edition, Cold Spring Harbor Laboratory, New York(2001) or in Ausubel et al., Current Protocols in Molecular Biology,John Wiley and Sons, Baltimore, Md. (1998), each of which isincorporated herein by reference.

A template nucleic acid can be obtained from a preparative method suchas genome isolation, genome fragmentation, gene cloning and/oramplification. The template can be obtained from an amplificationtechnique such as polymerase chain reaction (PCR), rolling circleamplification (RCA), multiple displacement amplification (MDA) or thelike. Exemplary methods for isolating, amplifying and fragmentingnucleic acids to produce templates for analysis on an array are setforth in U.S. Pat. No. 6,355,431 or 9,045,796, each of which isincorporated herein by reference. Amplification can also be carried outusing a method set forth in Sambrook et al., Molecular Cloning: ALaboratory Manual, 3rd edition, Cold Spring Harbor Laboratory, New York(2001) or in Ausubel et al., Current Protocols in Molecular Biology,John Wiley and Sons, Baltimore, Md. (1998), each of which isincorporated herein by reference.

Any of a variety of polymerases can be used in a method set forthherein. Reference to a particular polymerase, such as those exemplifiedthroughout this disclosure, will be understood to include functionalvariants thereof unless indicated otherwise. Particularly usefulfunctions of a polymerase include formation of a ternary complex orcatalysis of the polymerization of a nucleic acid strand using anexisting nucleic acid as a template. A particular polymerase activity orcharacteristic set forth herein, for example, forming ternary complexesthat are stabilized by a particular reagent such as Li⁺, PEI, betaineand/or an inhibitory metal ion such as Ca²⁺, can be shared bypolymerases that have been grouped by known classifications. Aparticularly useful classification is based on structural homology suchas the classification of polymerases into families identified as A, B,C, D, X, Y, and RT. DNA Polymerases in Family A include, for example, T7DNA polymerase, eukaryotic mitochondrial DNA Polymerase γ, E. coli DNAPol I, Thermus aquaticus Pol I, and Bacillus stearothermophilus Pol I.DNA Polymerases in Family B include, for example, eukaryotic DNApolymerases α, δ, and ε; DNA polymerase ζ; T4 DNA polymerase, Phi29 DNApolymerase, and RB69 bacteriophage DNA polymerase. Family C includes,for example, the E. coli DNA Polymerase III alpha subunit. Family Barchaeon DNA polymerases include, for example, Vent, Deep Vent, Pfu and9° N (e.g., Therminator™ DNA polymerase from New England BioLabs Inc.;Ipswich, Mass.) polymerases. Family D includes, for example, polymerasesderived from the Euryarchaeota subdomain of Archaea. DNA Polymerases inFamily X include, for example, eukaryotic polymerases Pol β, pol σ, Polλ, and Pol μ, and S. cerevisiae Pol4. DNA Polymerases in Family Yinclude, for example, Pol η, Pol ι, Pol κ, E. coli Pol IV (DINB) and E.coli Pol V (UmuD′2C). The RT (reverse transcriptase) family of DNApolymerases includes, for example, retrovirus reverse transcriptases andeukaryotic telomerases. Exemplary RNA polymerases include, but are notlimited to, viral RNA polymerases such as T7 RNA polymerase; EukaryoticRNA polymerases such as RNA polymerase I, RNA polymerase II, RNApolymerase III, RNA polymerase IV, and RNA polymerase V; and Archaea RNApolymerase.

The above classifications are provided for illustrative purposes. Itwill be understood that variations in the classification system arepossible. For example, in at least one classification system Family Cpolymerases have been categorized as a subcategory of Family X.Furthermore, polymerases can be classified according to othercharacteristics, whether functional or structural, that may or may notoverlap with the structural characteristics exemplified above. Someexemplary characteristics are set forth in further detail below.

Polymerases that may be used in a method or composition set forth hereininclude naturally occurring polymerases and modified variations thereof,including, but not limited to, mutants, recombinants, fusions, geneticmodifications, chemical modifications, synthetics, and analogs. Usefulpolymerases for ternary complex formation and detection are not limitedto polymerases that have the ability to catalyze a polymerizationreaction. Optionally, a useful polymerase will have the ability tocatalyze a polymerization reaction in at least one condition that is notused during formation or examination of a stabilized ternary complex.Optionally, a polymerase that participates in a stabilized ternarycomplex has modified properties, for example, enhanced binding affinityto nucleic acids, reduced binding affinity to nucleic acids, enhancedbinding affinity to nucleotides, reduced binding affinity tonucleotides, enhanced specificity for next correct nucleotides, reducedspecificity for next correct nucleotides, reduced catalysis rates,catalytic inactivity etc. Mutant polymerases include, for example,polymerases wherein one or more amino acids are replaced with otheramino acids, or insertions or deletions of one or more amino acids.Exemplary polymerases that can be used to form a stabilized ternarycomplex include, for example, wild type and mutant polymerases set forthin U.S. patent application Ser. No. 15/866,353, now published as US Pat.App. Pub. No. 2018/0155698 A1, or US Pat. App. Pub. No. 2017/0314072 A1,each of which is incorporated herein by reference.

Polymerases that contain an exogenous label moiety (e.g., an exogenousfluorophore), which can be used to detect the polymerase, can be usefulin some embodiments. Optionally, the label moiety can be attached afterthe polymerase has been at least partially purified using proteinisolation techniques. For example, the exogenous label moiety can bechemically linked to the polymerase using a free sulfhydryl or a freeamine moiety of the polymerase. This can involve chemical linkage to thepolymerase through the side chain of a cysteine residue, or through thefree amino group of the N-terminus. An exogenous label moiety can alsobe attached to a polymerase via protein fusion. Exemplary label moietiesthat can be attached via protein fusion include, for example, greenfluorescent protein (GFP), phycobiliproteins (e.g. phycocyanin andphycoerythrin) or wavelength-shifted variants of GFP orphycobiliproteins. In some embodiments, an exogenous label on apolymerase can function as a member of a FRET pair. The other member ofthe FRET pair can be an exogenous label that is attached to a nucleotidethat binds to the polymerase in a stabilized ternary complex. As such,the stabilized ternary complex can be detected or identified via FRET.

Alternatively, a polymerase that participates in a stabilized ternarycomplex need not be attached to an exogenous label. For example, thepolymerase need not be covalently attached to an exogenous label.Instead, the polymerase can lack any label until it associates with alabeled nucleotide and/or labeled nucleic acid (e.g. labeled primerand/or labeled template).

A ternary complex that is made or used in accordance with the presentdisclosure may optionally include one or more exogenous label(s). Thelabel can be attached to a component of the ternary complex (e.g.attached to the polymerase, template nucleic acid, primer and/or cognatenucleotide) prior to formation of the ternary complex. Exemplaryattachments include covalent attachments or non-covalent attachmentssuch as those set forth herein, in references cited herein or known inthe art. In some embodiments, a labeled component is delivered insolution to a solid support that is attached to an unlabeled component,whereby the label is recruited to the solid support by virtue of forminga stabilized ternary complex. As such, the support-attached componentcan be detected or identified based on observation of the recruitedlabel. Whether used in solution phase or on a solid support, exogenouslabels can be useful for detecting a stabilized ternary complex or anindividual component thereof, during an examination step. An exogenouslabel can remain attached to a component after the component dissociatesfrom other components that had formed a stabilized ternary complex.Exemplary labels, methods for attaching labels and methods for usinglabeled components are set forth in further detail below or in commonlyowned U.S. Pat. App. Pub. Nos. 2017/0022553 A1 or 2018/0044727 A1; orU.S. Pat. App. Ser. No. 15/851,383 (published as US Pat. App. Pub. No.2018/0187245 A1), Ser. No. 15/873,343 (published as US Pat. App. Pub.No. 2018/0208983 A1); or US Pat. App. Pub. No. 2018/0208983 A1, whichclaims priority to 62/450,397 and 62/506,759, each of which isincorporated herein by reference.

As set forth above, different activities of polymerases can be exploitedin a method set forth herein. A polymerase can be useful, for example,in one or both of an examination step or, as set forth in further detailbelow, in an extension step. The different activities can follow fromdifferences in the structure (e.g. via natural activities, mutations orchemical modifications). Nevertheless, polymerase can be obtained from avariety of known sources and applied in accordance with the teachingsset forth herein and recognized activities of polymerases. Useful DNApolymerases include, but are not limited to, bacterial DNA polymerases,eukaryotic DNA polymerases, archaeal DNA polymerases, viral DNApolymerases and phage DNA polymerases. Bacterial DNA polymerases includeE. coli DNA polymerases I, II and III, IV and V, the Klenow fragment ofE. coli DNA polymerase, Clostridium stercorarium (Cst) DNA polymerase,Clostridium thermocellum (Cth) DNA polymerase and Sulfolobussolfataricus (Sso) DNA polymerase. Eukaryotic DNA polymerases includeDNA polymerases α, β, γ, δ, €, η, ζ, λ, σ, μ, and k, as well as the Revlpolymerase (terminal deoxycytidyl transferase) and terminaldeoxynucleotidyl transferase (TdT). Viral DNA polymerases include T4 DNApolymerase, phi-29 DNA polymerase, GA-1, phi-29-like DNA polymerases,PZA DNA polymerase, phi-15 DNA polymerase, Cpl DNA polymerase, Cpl DNApolymerase, T7 DNA polymerase, and T4 polymerase. Other useful DNApolymerases include thermostable and/or thermophilic DNA polymerasessuch as Thermus aquaticus (Taq) DNA polymerase, Thermus filiformis (Tfi)DNA polymerase, Thermococcus zilligi (Tzi) DNA polymerase, Thermusthermophilus (Tth) DNA polymerase, Thermus flavusu (Tfl) DNA polymerase,Pyrococcus woesei (Pwo) DNA polymerase, Pyrococcus furiosus (Pfu) DNApolymerase and Turbo Pfu DNA polymerase, Thermococcus litoralis (Tli)DNA polymerase, Pyrococcus sp. GB-D polymerase, Thermotoga maritima(Tma) DNA polymerase, Bacillus stearothermophilus (Bst) DNA polymerase,Pyrococcus Kodakaraensis (KOD) DNA polymerase, Pfx DNA polymerase,Thermococcus sp. JDF-3 (JDF-3) DNA polymerase, Thermococcus gorgonarius(Tgo) DNA polymerase, Thermococcus acidophilium DNA polymerase;Sulfolobus acidocaldarius DNA polymerase; Thermococcus sp. go N-7 DNApolymerase; Pyrodictium occultum DNA polymerase; Methanococcus voltaeDNA polymerase; Methanococcus thermoautotrophicum DNA polymerase;Methanococcus jannaschii DNA polymerase; Desulfurococcus strain TOK DNApolymerase (D. Tok Pol); Pyrococcus abyssi DNA polymerase; Pyrococcushorikoshii DNA polymerase; Pyrococcus islandicum DNA polymerase;Thermococcus fumicolans DNA polymerase; Aeropyrum pernix DNA polymerase;and the heterodimeric DNA polymerase DP1/DP2. Engineered and modifiedpolymerases also are useful in connection with the disclosed techniques.For example, modified versions of the extremely thermophilic marinearchaea Thermococcus species 9° N (e.g., Therminator™ DNA polymerasefrom New England BioLabs Inc.; Ipswich, Mass.) can be used. Still otheruseful DNA polymerases, including the 3PDX polymerase are disclosed inU.S. Pat. No. 8,703,461, the disclosure of which is incorporated hereinby reference.

Useful RNA polymerases include, but are not limited to, viral RNApolymerases such as T7 RNA polymerase, T3 polymerase, SP6 polymerase,and Kll polymerase; Eukaryotic RNA polymerases such as RNA polymerase I,RNA polymerase II, RNA polymerase III, RNA polymerase IV, and RNApolymerase V; and Archaea RNA polymerase.

Another useful type of polymerase is a reverse transcriptase. Exemplaryreverse transcriptases include, but are not limited to, HIV-1 reversetranscriptase from human immunodeficiency virus type 1 (PDB 1HMV), HIV-2reverse transcriptase from human immunodeficiency virus type 2, M-MLVreverse transcriptase from the Moloney murine leukemia virus, AMVreverse transcriptase from the avian myeloblastosis virus, andTelomerase reverse transcriptase that maintains the telomeres ofeukaryotic chromosomes.

A polymerase having an intrinsic 3′-5′ proofreading exonuclease activitycan be useful for some embodiments. Polymerases that substantially lack3′-5′ proofreading exonuclease activity are also useful in someembodiments, for example, in most genotyping and sequencing embodiments.Absence of exonuclease activity can be a wild type characteristic or acharacteristic imparted by a variant or engineered polymerase structure.For example, exo minus Klenow fragment is a mutated version of Klenowfragment that lacks 3′-5′ proofreading exonuclease activity. Klenowfragment and its exo minus variant can be useful in a method orcomposition set forth herein.

A stabilized ternary complex can include a native nucleotide, nucleotideanalog or modified nucleotide as desired to suit a particularapplication or configuration of the methods. Optionally, a nucleotideanalog has a nitrogenous base, five-carbon sugar, and phosphate group,wherein any moiety of the nucleotide may be modified, removed and/orreplaced as compared to a native nucleotide. Nucleotide analogs may benon-incorporable nucleotides (i.e. nucleotides that are incapable ofreacting with the 3′ oxygen of a primer to form a covalent linkage).Such nucleotides that are incapable of incorporation include, forexample, monophosphate and diphosphate nucleotides. In another example,the nucleotide may contain modification(s) to the triphosphate groupthat render the nucleotide non-incorporable. Examples ofnon-incorporable nucleotides may be found in U.S. Pat. No. 7,482,120,which is incorporated by reference herein. In some embodiments,non-incorporable nucleotides may be subsequently modified to becomeincorporable. Non-incorporable nucleotide analogs include, but are notlimited to, alpha-phosphate modified nucleotides, alpha-beta nucleotideanalogs, beta-phosphate modified nucleotides, beta-gamma nucleotideanalogs, gamma-phosphate modified nucleotides, or caged nucleotides.Further examples of nucleotide analogs are described in U.S. Pat. No.8,071,755, which is incorporated by reference herein.

Nucleotide analogs that are used herein, for example, to participate instabilized ternary complexes can include terminators that reversiblyprevent subsequent nucleotide incorporation at the 3′-end of the primerafter the analog has been incorporated into the primer. For example,U.S. Pat. Nos. 7,544,794 and 8,034,923 (the disclosures of these patentsare incorporated herein by reference) describe reversible terminators inwhich the 3′-OH group is replaced by a 3′-ONH₂ moiety. Another type ofreversible terminator is linked to the nitrogenous base of a nucleotideas set forth, for example, in U.S. Pat. No. 8,808,989 (the disclosure ofwhich is incorporated herein by reference). Other reversible terminatorsthat similarly can be used in connection with the methods describedherein include those described in references cited elsewhere herein orin U.S. Pat. Nos. 7,956,171, 8,071,755, and 9,399,798 (the disclosuresof these U.S. patents are incorporated herein by reference). In certainembodiments, a reversible terminator moiety can be removed from aprimer, in a process known as “deblocking,” allowing for subsequentnucleotide incorporation. Compositions and methods for deblocking areset forth in references cited herein in the context of reversibleterminators.

Alternatively, nucleotide analogs irreversibly prevent nucleotideincorporation at the 3′-end of the primer to which they have beenincorporated. Irreversible nucleotide analogs include 2′,3′-dideoxynucleotides (ddNTPs such as ddGTP, ddATP, ddTTP, ddCTP).Dideoxynucleotides lack the 3′-OH group of dNTPs that would otherwiseparticipate in polymerase-mediated primer extension. Thus, the 3′position has a hydrogen moiety instead of the native hydroxyl moiety.Irreversibly terminated nucleotides can be particularly useful forgenotyping applications or other applications where primer extension orsequential detection along a template nucleic acid is not desired.

In particular embodiments, nucleotide analogs that are used herein, forexample, to participate in stabilized ternary complexes do not includeblocking groups (e.g. reversible terminators) that prevent subsequentnucleotide incorporation at the 3′-end of the primer after the analoghas been incorporated into the primer. This can be the case whether ornot an extension step is carried out using nucleotide(s) having ablocking group (e.g. reversible terminator).

In some embodiments, a nucleotide that is used herein, for example, toparticipate in forming a stabilized ternary complex, can include anexogenous label. For example, an exogenously labeled nucleotide caninclude a reversible or irreversible terminator moiety, an exogenouslylabeled nucleotide can be non-incorporable, an exogenously labelednucleotide can lack terminator moieties, an exogenously labelednucleotide can be incorporable or an exogenously labeled nucleotide canbe both incorporable and non-terminated. Exogenously labeled nucleotidescan be particularly useful when used to form a stabilized ternarycomplex with a non-labeled polymerase. Alternatively, an exogenous labelon a nucleotide can provide one partner in a fluorescence resonanceenergy transfer (FRET) pair and an exogenous label on a polymerase canprovide the second partner of the pair. As such, FRET detection can beused to identify a stabilized ternary complex that includes bothpartners.

Alternatively, a nucleotide that is used herein, for example, toparticipate in forming a ternary complex can lack exogenous labels (i.e.the nucleotide can be “non-labeled”). For example, a non-labelednucleotide can include a reversible or irreversible terminator moiety, anon-labeled nucleotide can be non-incorporable, a non-labeled nucleotidecan lack terminator moieties, a non-labeled nucleotide can beincorporable, or a non-labeled nucleotide can be both incorporable andnon-terminated. Non-labeled nucleotides can be useful when a label on apolymerase is used to detect a stabilized ternary complex or whenlabel-free detection is used. Non-labeled nucleotides can also be usefulin an extension step of a method set forth herein. It will be understoodthat absence of a moiety or function for a nucleotide refers to thenucleotide having no such function or moiety. However, it will also beunderstood that one or more of the functions or moieties set forthherein for a nucleotide, or analog thereof, or otherwise known in theart for a nucleotide, or analog thereof, can be specifically omitted ina method or composition set forth herein.

Optionally, a nucleotide (e.g. a native nucleotide or nucleotide analog)is present in a mixture during or after formation of a stabilizedternary complex. For example, at least 1, 2, 3, 4 or more nucleotidetypes can be present. Alternatively or additionally, at most 4, 3, 2, or1 nucleotide types can be present. Similarly, one or more nucleotidetypes that are present can be complementary to at least 1, 2, 3 or 4base types in a template nucleic acid. Alternatively or additionally,one or more nucleotide types that are present can be complementary to atmost 4, 3, 2, or 1 base types in a template nucleic acid. Different basetypes can be identifiable by the presence of different exogenous labelson the different nucleotides. Alternatively, two or more nucleotidetypes can have exogenous labels that are not distinguishable. In thelatter format the different nucleotides can nevertheless bedistinguished due to being separately delivered to a reaction vessel ordue to an encoding and decoding scheme as set forth, for example, inU.S. patent application Ser. No. 15/922,787, published as US Pat. App.Pub. No. 2018/0305749 A1, or U.S. Pat. No. 9,951,385, each of which isincorporated herein by reference.

Any nucleotide modification that stabilizes a polymerase in a ternarycomplex may be used in the methods disclosed herein. The nucleotide maybe bound permanently or transiently to a polymerase. Optionally, anucleotide analog is fused to a polymerase, for example, via a covalentlinker. Optionally, a plurality of nucleotide analogs is fused to aplurality of polymerases, wherein each nucleotide analog is fused to adifferent polymerase. Optionally, a nucleotide that is present in astabilized ternary complex is not the means by which the ternary complexis stabilized. Accordingly, any of a variety of other ternary complexstabilization methods may be combined in a reaction utilizing anucleotide analog.

In particular embodiments, the primer strand of a primed templatenucleic acid that is present in a stabilized ternary complex ischemically unchanged by the polymerase that is present during one ormore steps of a method set forth herein. For example, the primer neednot be extended by formation of a new phosphodiester bond, nor shortenedby nucleolytic degradation during a step for forming a stabilizedternary complex, nor during a step for detecting the stabilized ternarycomplex.

Particular embodiments of the methods set forth herein include a step offorming a mixture that includes several components. The components ofthe mixture can be delivered to a vessel in any desired order or theycan be delivered simultaneously. Furthermore, some of the components canbe mixed with each other to form a first mixture that is subsequentlycontacted with other components to form a more complex mixture. Takingas an example, a step of forming a mixture that includes a primedtemplate nucleic acid, a polymerase and a plurality of differentnucleotide types, it will be understood that the different nucleotidetypes in the plurality can be contacted with each other prior to beingcontacted with the primed template nucleic acid. Alternatively, two ormore of the nucleotide types can be delivered separately to the primedtemplate nucleic acid and/or the polymerase. As such, a first nucleotidetype can be contacted with the primed template nucleic acid prior tobeing contacted with a second nucleotide type. Alternatively oradditionally, the first nucleotide type can be contacted with thepolymerase prior to being contacted with a second nucleotide type.

In particular embodiments, polymerase and nucleotides are soluble in afluid that is used to stabilize ternary complex. For example, astabilizing fluid can advantageously be an aqueous fluid. Thestabilizing fluid can lack solvents that do not solubilize polymeraseand/or nucleotides. For example, a ternary complex stabilizing fluid canbe devoid of alcohols or oils that do not solubilize polymerase and/ornucleotides. Exemplary fluids that do not solubilize polymerase and/ornucleotides are set forth in U.S. patent application Ser. No.16/164,417, which claims priority to U.S. Pat. App. Ser. No. 62/574,308,each of which is incorporated herein by reference. In some embodiments,a ternary complex stabilizing fluid can function as a wash fluid toremove non-complexed nucleotide or polymerase from an immobilizedternary complex.

A ternary complex stabilizing fluid can be devoid of one or morecomponents of a ternary complex binding reaction prior to beingcontacted with a ternary complex. For example, a stabilizing fluid thatcontains Li⁺, PEI, betaine or an inhibitory metal cation (e.g. Ca²⁺) canbe devoid of polymerase or nucleotides prior to being contacted with aternary complex. As such, the stabilizing fluid can function as a washto remove excess polymerase or nucleotides from a binding reaction thatpreviously functioned to form a ternary complex. In particularembodiments, the concentration of ternary complex in a stabilizationfluid is greater than the concentration of free nucleotide and/orpolymerase in the fluid. The free nucleotide and polymerase in thisfluid can be the same type that is present in the ternary complex. Itwill be understood that Li⁺, PEI, betaine, inhibitory metal cation (e.g.Ca²⁺) or other ternary complex stabilizing agent can be present duringternary complex formation. Alternatively, the ternary complexstabilizing agent can be introduced to a ternary complex that hasalready been formed.

A ternary complex can be detected via the presence of an exogenous labelon one or more component of the complex. Examples of useful exogenouslabels include, but are not limited to, radiolabel moieties, luminophoremoieties, fluorophore moieties, quantum dot moieties, chromophoremoieties, enzyme moieties, electromagnetic spin labeled moieties,nanoparticle light scattering moieties, and any of a variety of othersignal generating moieties known in the art. Suitable enzyme moietiesinclude, for example, horseradish peroxidase, alkaline phosphatase,beta-galactosidase, or acetylcholinesterase. Exemplary fluorophoremoieties include, but are not limited to rhodols; resorufins; coumarins;xanthenes; acridines; fluoresceins; rhodamines; erythrins; cyanins;phthalaldehydes; naphthylamines; fluorescamines; benzoxadiazoles;stilbenes; pyrenes; indoles; borapolyazaindacenes; quinazolinones;eosin; erythrosin; Malachite green; CY dyes (GE Biosciences), includingCy3 (and its derivatives) and Cy5 (and its derivatives); DYOMICS andDYLIGHT dyes (Dyomics) including DY-547, DY-630, DY-631, DY-632, DY-633,DY-634, DY-635, DY-647, DY-649, DY-652, DY-678, DY-680, DY-682, DY-701,DY-734, DY-752, DY-777 and DY-782; Lucifer Yellow; CASCADE BLUE; TEXASRED; BODIPY (boron-dipyrromethene) (Molecular Probes) dyes includingBODIPY 630/650 and BODIPY 650/670; ATTO dyes (Atto-Tec) including ATTO390, ATTO 425, ATTO 465, ATTO 610 611X, ATTO 610, ATTO 635; ALEXA FLUORSincluding ALEXA FLUOR 633, ALEXA FLUOR 647, ALEXA FLUOR 660, ALEXA FLUOR700, ALEXA FLUOR 750, and ALEXA FLUOR 680 (Molecular Probes); DDAO(7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one or any derivativesthereof) (Molecular Probes); QUASAR dyes (Biosearch); IRDYES dyes(LiCor) including IRDYE 700DX (NHS ester), IRDYE 800RS (NHS ester) andIRDYE 800CW (NHS ester); EVOBLUE dyes (Evotech Biosystems); JODA 4 dyes(Applied Biosystems); HILYTE dyes (AnaSpec); MR121 and MR200 dyes(Roche); Hoechst dyes 33258 and 33242 (Invitrogen); FAIR OAKS RED(Molecular Devices); SUNNYVALE RED (Molecular Devices); LIGHT CYCLER RED(Roche); EPOCH (Glen Research) dyes including EPOCH REDMOND RED, EPOCHYAKIMA YELLOW, EPOCH GIG HARBOR GREEN; Tokyo green (M. Kamiya, et al.,2005 Angew. Chem. Int. Ed. 44:5439-5441); and CF dyes including CF 647and CF555 (Biotium), and others known in the art such as those describedin Principles of Fluorescence Spectroscopy, Joseph R. Lakowicz (Editor),Plenum Pub Corp, 2nd edition (July 1999) and the 6th Edition ofMolecular Probes Handbook by Richard P. Hoagland.

A secondary label can be used in a method of the present disclosure. Asecondary label is a binding moiety that can bind specifically to apartner moiety. For example, a ligand moiety can be attached to apolymerase, nucleic acid or nucleotide to allow detection via specificaffinity for labeled receptor. Exemplary pairs of binding moieties thatcan be used include, without limitation, antigen and immunoglobulin oractive fragments thereof, such as FAbs; immunoglobulin andimmunoglobulin (or active fragments, respectively); avidin and biotin,or analogs thereof having specificity for avidin; streptavidin andbiotin, or analogs thereof having specificity for streptavidin; orcarbohydrates and lectins.

In some embodiments, the secondary label can be a chemically modifiablemoiety. In this embodiment, labels having reactive functional groups canbe incorporated into a stabilized ternary complex. Subsequently, thefunctional group can be covalently reacted with a primary label moiety.Suitable functional groups include, but are not limited to, aminogroups, carboxy groups, maleimide groups, oxo groups, groups thatparticipate in click reactions and thiol groups.

A label can be attached to a nucleotide, polymerase or other moleculevia a linker. A linker that is present in a nucleotide or polymerase canbe, but need not be, cleavable. For example, the linker can be stable toconditions used in methods set forth herein such that the covalentstructure of the linker is not changed during any particular step, orthroughout all steps, of a method set forth herein. A linker that ispresent in a nucleotide analog can be at least as chemically stable asone or more other moieties in the analog. For example, the linker can beas chemically stable as the nitrogenous base, sugar and/or phosphatemoiety during any particular step, or throughout all steps, of a methodset forth herein.

In alternative embodiments, a ternary complex can lack exogenous labels.For example, a stabilized ternary complex and all componentsparticipating in the stabilized ternary complex (e.g. polymerase,template nucleic acid, primer and/or cognate nucleotide) can lack one,several or all of the exogenous labels described herein or in thereferences that are cited and incorporated herein. In such embodiments,ternary complexes can be detected based on intrinsic properties of thestabilized ternary complex, such as mass, charge, intrinsic opticalproperties or the like. Exemplary methods for detecting non-labeledternary complexes are set forth in commonly owned U.S. Pat. App. Pub.No. 2017/0022553 A1 PCT App. Ser. No. PCT/US16/68916 (published as WO2017/117243 A1), or U.S. Pat. App. Ser. Nos. 62/375,379 (now publishedas US Pat. App. Pub. No. 2018/0044727 A1 or Ser. No. 15/677,870,published as US Pat. App. Pub. No. 2018/0044715 A1, each of which isincorporated herein by reference.

Generally, detection can be achieved in an examination step by methodsthat perceive a property that is intrinsic to a ternary complex or alabel moiety attached thereto. Exemplary properties upon which detectioncan be based include, but are not limited to, mass, electricalconductivity, energy absorbance, luminescence (e.g. fluorescence) or thelike. Detection of luminescence can be carried out using methods knownin the art pertaining to nucleic acid arrays. A luminophore can bedetected based on any of a variety of luminescence properties including,for example, emission wavelength, excitation wavelength, fluorescenceresonance energy transfer (FRET) intensity, quenching, anisotropy orlifetime. Other detection techniques that can be used in a method setforth herein include, for example, mass spectrometry which can be usedto perceive mass; surface plasmon resonance which can be used toperceive binding to a surface; absorbance which can be used to perceivethe wavelength of the energy a label absorbs; calorimetry which can beused to perceive changes in temperature due to presence of a label;electrical conductance or impedance which can be used to perceiveelectrical properties of a label, or other known analytic techniques.Examples of reagents and conditions that can be used to create,manipulate and detect stabilized ternary complexes include, for example,those set forth in commonly owned U.S. Pat. App. Pub. No. 2017/0022553A1; PCT App. Ser. No. PCT/US16/68916, published as WO 2017/117243 A1; orU.S. patent application Ser. No. 15/677,870, published as US Pat. App.Pub. No. 2018/0044715 A1; Ser. No. 15/851,383, published as US Pat. App.Pub. No. 2018/0187245 A1; Ser. No. 15/873,343, published as US Pat. App.Pub. No. 2018/0208983 A1; US Pat. App. Pub. No. 2018/0208983 A1, whichclaims priority to 62/450,397 and 62/506,759, each of which isincorporated herein by reference.

Detection of ternary complexes can take place in different types ofreaction mixtures, but generally takes place under conditions thatstabilize ternary complexes. The conditions can also be selected toreduce the concentration of labeled species (e.g., labeled nucleotides)below the level that was used to form the ternary complex. Optionally,an examination step, or sub-step, involves detecting the interaction ofa polymerase and cognate nucleotide with a primed template nucleic acidin the absence of non-complexed labeled nucleotide and polymerase.Optionally, the examination step or sub-step involves detecting alabeled nucleotide in a stabilized ternary complex after non-boundnucleotides have been removed from contact with the ternary complexusing a stabilizing fluid. The formation of a ternary complex may bedetected or monitored by detecting or monitoring a label attached to thenucleotide that participates in the ternary complex. Optionally, theabsence of formation of ternary complex is detected or monitored.Optionally, the dissociation of a ternary complex is monitored.

A step for detecting ternary complexes can take place during a wash stepcarried out using a ternary complex stabilizing fluid. The stabilizingfluid can contain Li⁺, PEI, betaine or an inhibitory metal cation (e.g.Ca²⁺). Optionally, the stabilizing fluid is held static (i.e., notmoving or flowing) during the detection step. However, the fluid canflow, for example, through a flow cell that contains a ternary complexthat is to be detected. Advantageously, detection of ternary complexesduring a wash step can reduce background signal associated withnon-bound nucleotides or polymerases that may harbor detectable labels.Again, by this approach ternary complex detection can take place in areaction mixture different from the reaction mixture that provided thelabeled nucleotide(s) to the primed template nucleic acid molecule,which optionally can be blocked at its 3′-end with a reversibleterminator moiety.

Examination and detection of a stabilized ternary complex may beaccomplished in different ways. For example, monitoring can includemeasuring association kinetics for the interaction between two or moreof the components of the complex. Monitoring the interaction can includemeasuring equilibrium binding signals or equilibrium binding constants.Thus, for example, the monitoring may include measuring equilibriumbinding signals, or the equilibrium binding constant in the presence ofone or more of the labeled nucleotides. Monitoring the interaction caninclude, for example, measuring dissociation kinetics of the nucleotidefrom the primed template nucleic acid in the presence of any one of thefour nucleotides. Optionally, monitoring the interaction of thepolymerase with the primed template nucleic acid molecule in thepresence of a nucleotide molecule includes measuring the kinetics of thedissociation of the closed complex. Techniques for measuringassociation, equilibrium and dissociation kinetics are known and can bereadily modified for use in a method set forth herein by one in the art.See, for example, Markiewicz et al., Nucleic Acids Research40(16):7975-84 (2012); Xia et al., J. Am. Chem. Soc. 135(1):193-202(2013); Brown et al., J. Nucleic Acids, Article ID 871939, 11 pages(2010); Washington, et al., Mol. Cell. Biol. 24(2):936-43 (2004); Walshand Beuning, J. Nucleic Acids, Article ID 530963, 17 pages (2012); andRoettger, et al., Biochemistry 47(37):9718-9727 (2008), which areincorporated by reference herein. It will be understood that a detectiontechnique can accumulate signal over a relatively brief duration as istypically understood to be a single timepoint acquisition.Alternatively, signal can be continuously monitored over time as istypical of a time-based acquisition. It is also possible to acquire aseries of timepoints in a periodic fashion to obtain a time-basedacquisition.

Some embodiments of the methods set forth herein utilize two or moredistinguishable signals to distinguish stabilized ternary complexes fromeach other and/or to distinguish one base type in a template nucleicacid from another base type. For example, two or more luminophores canbe distinguished from each other based on unique optical properties suchas unique wavelength for excitation or unique wavelength of emission. Inparticular embodiments, a method can distinguish different stabilizedternary complexes based on differences in luminescence intensity. Forexample, a first ternary complex can be detected in a condition where itemits less intensity than a second ternary complex. Such intensityscaling (sometimes called ‘grey scaling’) can exploit anydistinguishable intensity difference. Exemplary differences include aparticular stabilized ternary complex having an intensity that is 10%,25%, 33%, 50%, 66%, or 75% compared to the intensity of anotherstabilized ternary complex that is to be detected.

Intensity differences can result from use of different luminophores eachhaving a different extinction coefficient (i.e. resulting in differentexcitation properties) and/or different luminescence quantum yield (i.e.resulting in different emission properties). Alternatively, the sameluminophore type can be used but can be present in different amounts.For example, all members of a first population of ternary complexes canbe labeled with a particular luminophore, whereas a second populationhas only half of its members labeled with the luminophore. In thisexample, the second population would be expected to produce half thesignal of the first population. The second population can be produced,for example, by using a mixture of labeled nucleotides and unlabelednucleotides (in contrast to the first population containing primarilylabeled nucleotides). Similarly, the second population can be produced,for example, by using a mixture of labeled polymerases and unlabeledpolymerases (in contrast to the first population containing primarilylabeled polymerases). In an alternative labeling scheme, a firstpopulation of ternary complexes can include polymerase molecules thathave multiple labels that produce a particular luminescent signal and asecond population of ternary complexes can include polymerase moleculesthat each have only one of the labels that produces the luminescentsignal.

In some embodiments, the examination step is carried out in a way thatthe identity of at least one nucleotide type is imputed, for example, asset forth in commonly owned U.S. Pat. No. 9,951,385 or U.S. patentapplication Ser. No. 15/922,787, published as US Pat. App. Pub. No.2018/0305749 A1, each of which is incorporated herein by reference. Forexample, an examination step can include steps of (a) providing a fluidthat includes a primed template nucleic acid, a polymerase andnucleotide cognates of first, second and third base types in thetemplate under ternary complex stabilizing conditions; (b) optionallyremoving non-bound polymerase and non-bound nucleotides from the fluid;(c) examining the fluid to determine whether a ternary complex formed,wherein the fluid includes Li⁺, PEI, betaine and/or an inhibitory metalcation (e.g. Ca²⁺); and (d) identifying the next correct nucleotide forthe primed template nucleic acid molecule, wherein the next correctnucleotide is identified as a cognate of the first, second or third basetype if ternary complex is detected in step (c), and wherein the nextcorrect nucleotide is imputed to be a nucleotide cognate of a fourthbase type based on the absence of a ternary complex in step (c).

Alternatively or additionally to using imputation, an examination stepcan use a decoding or disambiguation scheme to identify one or morenucleotide types, for example, as set forth in commonly owned U.S. Pat.No. 9,951,385 or U.S. patent application Ser. No. 15/922,787, publishedas US Pat. App. Pub. No. 2018/0305749 A1, each of which is incorporatedherein by reference. For example, examination can be carried out by (a)contacting a primed template nucleic acid with a polymerase and a firstmixture of nucleotides under ternary complex stabilizing conditions,wherein the first mixture includes a nucleotide cognate of a first basetype and a nucleotide cognate of a second base type; (b) contacting theprimed template nucleic acid with a polymerase and a second mixture ofnucleotides under ternary complex stabilizing conditions, wherein thesecond mixture includes a nucleotide cognate of the first base type anda nucleotide cognate of a third base type; (c) examining products ofsteps (a) and (b) for signals produced by a ternary complex thatincludes the primed template nucleic acid, a polymerase and a nextcorrect nucleotide, wherein signals acquired for the product of step (a)are ambiguous for the first and second base type, and wherein signalsacquired for the product of step (b) are ambiguous for the first andthird base type, wherein the products of steps (a) and (b) are examinedin the presence of Li⁺, PEI, betaine and/or an inhibitory metal cation(e.g. Ca²⁺); (d) disambiguating signals acquired in step (c) to identifya base type that binds the next correct nucleotide. Optionally, toachieve disambiguation (i) the first base type is correlated withpresence of signals for the product of step (a) and presence of signalsfor the product of step (b), (ii) the second base type is correlatedwith presence of signals for the product of step (a) and absence ofsignals for the product of step (b), and (iii) the third base type iscorrelated with absence of signals for the product of step (a) andpresence of signals for the product of step (b). Optionally, non-boundpolymerase and/or non-bound nucleotide are removed from the products ofsteps (a) and (b) prior to step (c).

The disclosed techniques provide advantages over a variety of othertechniques used in the DNA sequencing field. For example, there is norequirement for a label (e.g., a FRET partner) to be present on apolymerase or primed template nucleic acid. Indeed, in certainembodiments the polymerase is unlabeled, or does not generate any signalused for identifying cognate or non-cognate nucleotide in a ternarycomplex. Signal energy (e.g. FRET) need not transfer between apolymerase and a labeled nucleotide to render the ternary complexdetectable in a method of the present disclosure. A label or dye of adetectable nucleotides employed in a method herein need not be anintercalating dye (e.g., not an intercalating dye disclosed in U.S. Pat.No. 8,399,196), that changes its signal-generating properties (e.g.,fluorescent output) upon participating in a ternary complex. As well,the label or dye present on a labeled nucleotide need not be aconformationally sensitive dye that changes spectral properties when itis the cognate nucleotide present in a ternary complex.

In some embodiments, a method of the present disclosure can include astep of extending a primer. For example, a method of detecting a primedtemplate nucleic acid can include the steps of: (a) providing a mixturethat includes a ternary complex, the ternary complex including a primedtemplate nucleic acid, a polymerase, and a nucleotide cognate for thenext base of the primed template nucleic acid, wherein the mixturefurther includes excess polymerase of the same type present in theternary complex and excess nucleotide of the same type present in theternary complex; (b) replacing the excess polymerase and the excessnucleotide with a fluid containing Li⁺, PEI, betaine and/or aninhibitory metal cation (e.g. Ca²⁺); (c) detecting the ternary complexwhile it is in contact with the fluid containing the stabilizing agent;(d) identifying the next correct base for the primed template nucleicacid molecule from the result of step (c); and (e) extending the primerof primed template nucleic acid. Optionally, the method can furtherinclude a step of (f) repeating steps (a) through (e) using the primedtemplate nucleic acid having the extended primer in place of the primedtemplate nucleic acid.

In yet another example of a method that includes a primer extensionstep, the steps of the method can include: (a) providing a mixture thatincludes a ternary complex, the ternary complex including a primedtemplate nucleic acid, a polymerase, and a nucleotide cognate for thenext base of the primed template nucleic acid, wherein the mixturefurther includes excess nucleotide of the same type present in theternary complex; (b) replacing the excess nucleotide with a fluidcontaining Li⁺, PEI, betaine and/or an inhibitory metal cation (e.g.Ca²⁺) (c) detecting the ternary complex while it is in contact with thefluid containing Li⁺, PEI, betaine and/or an inhibitory metal cation(e.g. Ca²⁺) (d) identifying the next correct base for the primedtemplate nucleic acid molecule from the result of step (c); and (e)extending the primer of primed template nucleic acid. Optionally, themethod can further include a step of (f) repeating steps (a) through (e)using the primed template nucleic acid having the extended primer inplace of the primed template nucleic acid.

A primer extension step can be carried out by contacting a primedtemplate nucleic acid with an extension reaction mixture. Typically,fluid that was present in a previous examination step is removed andreplaced with the extension reaction mixture. Alternatively, theextension reaction mixture can be formed by adding one or more reagentsto the fluid that was present in the examination step. Optionally, theextension reaction mixture includes a different composition ofnucleotides than an examination step. For example, an examination stepcan include one or more nucleotide types that are not present in theextension reaction and vice versa. By way of more specific example, anextension step can omit at least one type of nucleotide and anexamination step can employ at least four types of nucleotides.Optionally, one or more nucleotide types is added to an examinationmixture for a primer extension step.

Nucleotides present in an examination step may cause unwanted nucleotideincorporation if carried over into an extension step. Thus, a wash stepcan be employed prior to a primer extension step to remove nucleotides.Optionally, free nucleotides may be removed by enzymes such asphosphatases, by chemical modification or by physical separationtechniques.

A primer extension step can use any of a variety of polymerasesincluding, for example, a polymerase set forth above in the context offorming a ternary complex. However, the polymerase used for extensionwill be catalytically active and used in a condition that does notpreclude catalysis. A polymerase that is used for an extension step neednot be attached to an exogenous label (e.g. covalently or otherwise).Alternatively, a polymerase that is used for primer extension caninclude an exogenous label, for example, a label that was used in aprevious examination step. Ternary complex formation and extension stepscan use the same type of polymerase, albeit in different conditions.Alternatively, the two steps can be carried out by different types ofpolymerases.

Adding a reversibly terminated nucleotide to the 3′ end of a primerprovides a means to prevent more than one nucleotide from being added tothe primer during the extension step and further prevents unwantedextension of the primer in a subsequent examination step. Thus, aposition in a template that is adjacent to a nucleotide of a particulartype can be examined. In such embodiments, a stabilized ternary complexcan be formed at the position and examined to detect the next correctnucleotide for the template that is hybridized to the extended,reversibly terminated primer. The method can be repeated in a step-wisefashion by then removing or modifying the reversible terminator moietyfrom the extended, reversibly terminated primer to produce an extendibleprimer.

Typically, a reversibly terminated nucleotide that is added to a primerin a method set forth herein does not have an exogenous label. This isbecause the extended primer need not be detected in a method set forthherein. However, if desired, one or more types of reversibly terminatednucleotides used in a method set forth herein can be detected, forexample, via exogenous labels attached to the nucleotides. Exemplaryreversible terminator moieties, methods for incorporating them intoprimers and methods for modifying the primers for further extension(often referred to as ‘deblocking’) are set forth in U.S. Pat. Nos.7,544,794; 7,956,171; 8,034,923; 8,071,755; 8,808,989; or 9,399,798.Further examples are set forth in Bentley et al., Nature 456:53-59(2008), WO 04/018497; U.S. Pat. No. 7,057,026; WO 91/06678; WO07/123744; U.S. Pat. Nos. 7,329,492; 7,211,414; 7,315,019; 7,405,281,and US 2008/0108082, each of which is incorporated herein by reference.Further examples of reagents and conditions that can be used for apolymerase-based primer extension step include, for example, those setforth in commonly owned U.S. Pat. App. Pub. No. 2017/0022553 A1 or U.S.patent application Ser. No. 15/677,870, published as US Pat. App. Pub.No. 2018/0044727 A1; Ser. No. 15/581,383, published as US Pat. App. Pub.No. 2018/0208922 A1; or US Pat. App. Pub. No. 2018/0208983 A1, whichclaims priority to 62/450,397 and 62/506,759, each of which isincorporated herein by reference.

A method of the present disclosure can include a step of contacting aprimed template nucleic acid with a polymerase and a mixture ofnucleotides under conditions to produce an extended primer hybrid. Oneor more of the nucleotides in the mixture can be reversibly terminated.For example, at least 1, 2, 3, 4 or more nucleotide types in the mixturecan be reversibly terminated. Alternatively or additionally, at most 4,3, 2, or 1 nucleotide types in the mixture can be reversibly terminated.Similarly, one or more nucleotide types that are reversibly terminatedin the mixture can be complementary to at least 1, 2, 3 or 4 base typesin a template nucleic acid. Alternatively or additionally, thereversibly terminated nucleotide types in the mixture can becomplementary to at most 4, 3, 2, or 1 base types in a template nucleicacid. Reversibly terminated and non-terminated nucleotides can bepresent simultaneously in an extension reaction. For example, some orall of the nucleotide types can be delivered simultaneously in a singleextension reaction. Alternatively, different nucleotide types can beserially delivered (individually or in subsets) such that they arecombined into a single extension reaction. Use of a mixture ofterminated and extendible nucleotides can allow for determination of alow-resolution sequence and/or to extend read lengths as set forth, forexample, in U.S. patent application Ser. No. 16/265,942, which claimspriority to U.S. Pat. App. Ser. No. 62/626,836, each of which isincorporated herein by reference.

In particular embodiments, reagents that are used during a primerextension step are removed from contact with the primed template nucleicacid prior to a step of forming a stabilized ternary complex with theprimed template nucleic acid. For example, removal of a nucleotidemixture that was used for an extension step can be desirable when one ormore types of nucleotides in the mixture would interfere with formationor detection of a ternary complex in a subsequent examination step.Similarly, it may be desirable to remove polymerases or cofactors thatwere used in a primer extension step to prevent unwanted catalyticactivity during the examination step. Removal can be followed by a washstep, wherein an inert fluid is used to purge the primed templatenucleic acid of residual components of the extension mixture.

Wash steps can be performed between any of a variety of steps set forthherein. For example, a wash step can be useful for separating a primedtemplate nucleic acid from other reagents that were contacted with theprimed template nucleic acid under ternary complex stabilizingconditions. Such a wash can remove one or more reagents from interferingwith examination of a mixture or from contaminating a second mixturethat is to be formed on a substrate (or in a vessel) that had previouslybeen in contact with the first mixture. For example, a primed templatenucleic acid can be contacted with a polymerase and at least onenucleotide type to form a first mixture under ternary complexstabilizing conditions, and the first mixture can be examined.Optionally, a wash can be carried out prior to examination to removereagents that are not participating in formation of a stabilized ternarycomplex. Alternatively or additionally, a wash can be carried out afterthe examination step to remove one or more component of the firstmixture from the primed template nucleic acid. Then the primed templatenucleic acid can be contacted with a polymerase and at least one othernucleotide to form a second mixture under ternary complex stabilizingconditions, and the second mixture can be examined for ternary complexformation. As before, an optional wash can be carried out prior to thesecond examination to remove reagents that are not participating information of a stabilized ternary complex. Washes that are contactedwith a ternary complex can include a stabilizing fluid such as thosethat include Li⁺, PEI, betaine, inhibitory metal ion (e.g. Ca²⁺) orother ternary complex stabilizing agents set forth herein.

A method of the present disclosure can include multiple repetitions ofsteps set forth herein. Such repetition can provide a sequence for atemplate nucleic acid or a signature for the template nucleic acid.Examination and extension steps can be repeated multiple times as canoptional steps of deblocking primers or washing away unwanted reactantsor products between various steps. Accordingly, a primed templatenucleic acid can be subjected at least 2, 5, 10, 25, 50, 100 or moresteps of a method set forth herein. Not all of the steps need to berepeated nor do repeated steps need to occur in the same order in eachrepetition. For example, next correct nucleotides at each position of atemplate can be identified using real time analysis (i.e. in parallelwith fluidic and detection steps of a sequencing method). However, realtime analysis is not necessary and instead next correct nucleotides canbe identified after some or all of the fluidic and detection steps havebeen completed.

A stabilized ternary complex, or a component that is capable of forming(i.e. participating in the formation of) a stabilized ternary complex,can be attached to a solid support. The solid support can be made fromany of a variety of materials used for analytical biochemistry. Suitablematerials may include glass, polymeric materials, silicon, quartz (fusedsilica), borofloat glass, silica, silica-based materials, carbon,metals, an optical fiber or bundle of optical fibers, sapphire, orplastic materials. The particular material can be selected based onproperties desired for a particular use. For example, materials that aretransparent to a desired wavelength of radiation are useful foranalytical techniques that will utilize radiation of that wavelength.Conversely, it may be desirable to select a material that does not passradiation of a certain wavelength (e.g. being opaque, absorptive orreflective). Other properties of a material that can be exploited areinertness or reactivity to certain reagents used in a downstreamprocess, such as those set forth herein, or ease of manipulation, or lowcost of manufacture.

A particularly useful solid support is a particle such as a bead ormicrosphere. Populations of beads can be used for attachment ofpopulations of stabilized ternary complexes or components capable offorming the complexes (e.g. polymerases, templates, primers ornucleotides). In some embodiments, it may be useful to use aconfiguration whereby each bead has a single type of stabilized ternarycomplex or a single type of component capable of forming the complex.For example, an individual bead can be attached to a single type ofternary complex, a single type of template allele, a single type oftemplate locus, a single type of allele-specific primer, a single typeof locus-specific primer or a single type of nucleotide. Alternatively,different types of components need not be separated on a bead-by-beadbasis. As such, a single bead can bear multiple different types ofternary complexes, template nucleic acids, primers, primed templatenucleic acids and/or nucleotides. The composition of a bead can vary,depending for example, on the format, chemistry and/or method ofattachment to be used. Exemplary bead compositions include solidsupports, and chemical functionalities imparted thereto, used in proteinand nucleic acid capture methods. Such compositions include, forexample, plastics, ceramics, glass, polystyrene, melamine,methylstyrene, acrylic polymers, paramagnetic materials, thoria sol,carbon graphite, titanium dioxide, latex or cross-linked dextrans suchas Sepharose™, cellulose, nylon, cross-linked micelles and Teflon™, aswell as other materials set forth in “Microsphere Detection Guide” fromBangs Laboratories, Fishers Ind., which is incorporated herein byreference.

The geometry of a particle, bead or microsphere also can correspond to awide variety of different forms and shapes. For example, they can besymmetrically shaped (e.g. spherical or cylindrical) or irregularlyshaped (e.g. controlled pore glass). In addition, beads can be porous,thus increasing the surface area available for capture of ternarycomplexes or components thereof. Exemplary sizes for beads used hereincan range from nanometers to millimeters or from about 10 nm-1 mm.

In particular embodiments, beads can be arrayed or otherwise spatiallydistinguished. Exemplary bead-based arrays that can be used include,without limitation, a BeadChip′ Array available from Illumina, Inc. (SanDiego, Calif.) or arrays such as those described in U.S. Pat. Nos.6,266,459; 6,355,431; 6,770,441; 6,859,570; or 7,622,294; or PCTPublication No. WO 00/63437, each of which is incorporated herein byreference. Beads can be located at discrete locations, such as wells, ona solid-phase support, whereby each location accommodates a single bead.Alternatively, discrete locations where beads reside can each include aplurality of beads as described, for example, in U.S. Pat. App. Pub.Nos. 2004/0263923 A1, 2004/0233485 A1, 2004/0132205 A1, or 2004/0125424A1, each of which is incorporated herein by reference.

As will be recognized from the above bead array embodiments, a method ofthe present disclosure can be carried out in a multiplex format wherebymultiple different types of nucleic acids are detected in parallel in amethod set forth herein. Although it is also possible to seriallyprocess different types of nucleic acids using one or more steps of themethods set forth herein, parallel processing can provide cost savings,time savings and uniformity of conditions. A composition or method ofthe present disclosure can include at least 2, 10, 100, 1×10³, 1×10⁴,1×10⁵, 1×10⁶, 1×10⁹, or more different nucleic acids. Alternatively oradditionally, a composition or method of the present disclosure caninclude at most 1×10⁹, 1×10⁶, 1×10⁵, 1×10⁴, 1×10³, 100, 10, 2 or fewer,different nucleic acids. Accordingly, various reagents or products setforth herein as being useful in the apparatus or methods (e.g. primedtemplate nucleic acid or stabilized ternary complexes) can bemultiplexed to have different types or species in these ranges.

Further examples of commercially available arrays that can be usedinclude, for example, an Affymetrix GeneChip™ array. A spotted array canalso be used according to some embodiments. An exemplary spotted arrayis a CodeLink™ Array available from Amersham Biosciences. Another arraythat is useful is one that is manufactured using inkjet printing methodssuch as SurePrint™ Technology available from Agilent Technologies.

Other useful arrays include those that are used in nucleic acidsequencing applications. For example, arrays that are used to immobilizeamplicons of genomic fragments (often referred to as clusters) can beparticularly useful. Examples of nucleic acid sequencing arrays that canbe used herein include those described in Bentley et al., Nature456:53-59 (2008), PCT Pub. Nos. WO 91/06678; WO 04/018497 or WO07/123744; U.S. Pat. Nos. 7,057,026; 7,211,414; 7,315,019; 7,329,492 or7,405,281; or U.S. Pat. App. Pub. No. 2008/0108082, each of which isincorporated herein by reference.

A nucleic acid can be attached to a support in a way that providesdetection at a single molecule level or at an ensemble level. Forexample, a plurality of different nucleic acids can be attached to asolid support in a way that an individual stabilized ternary complexthat forms on one nucleic acid molecule on the support can bedistinguished from all neighboring ternary complexes that form on thenucleic acid molecules of the support. As such, one or more differenttemplates can be attached to a solid support in a format where eachsingle molecule template is physically isolated and detected in a waythat the single molecule is resolved from all other molecules on thesolid support.

Alternatively, a method of the present disclosure can be carried out forone or more nucleic acid ensembles, an ensemble being a population ofnucleotides having a common template sequence. Cluster methods can beused to attach one or more ensembles to a solid support. As such, anarray can have a plurality of ensembles, each of the ensembles beingreferred to as a cluster or array feature in that format. Clusters canbe formed using methods known in the art such as bridge amplification oremulsion PCR. Useful bridge amplification methods are described, forexample, in U.S. Pat. No. 5,641,658 or 7,115,400; or U.S. Patent Pub.Nos. 2002/0055100 A1; 2004/0002090 A1; 2004/0096853 A1; 2007/0128624 A1;or 2008/0009420 A1. Emulsion PCR methods include, for example, methodsdescribed in Dressman et al., Proc. Natl. Acad. Sci. USA 100:8817-8822(2003), WO 05/010145, or U.S. Patent Pub. Nos. 2005/0130173 A1 or2005/0064460 A1, each of which is incorporated herein by reference inits entirety. Another useful method for amplifying nucleic acids on asurface to form clusters is rolling circle amplification (RCA), forexample, as described in Lizardi et al., Nat. Genet. 19:225-232 (1998)or US 2007/0099208 A1, each of which is incorporated herein byreference.

In particular embodiments, a stabilized ternary complex, polymerase,nucleic acid or nucleotide is attached to a flow cell surface or to asolid support in a flow cell. A flow cell allows convenient fluidicmanipulation by passing solutions into and out of a fluidic chamber thatcontacts the support-bound, ternary complex. The flow cell also providesfor detection of the fluidically manipulated components. For example, adetector can be positioned to detect signals from the solid support,such as signals from a label that is recruited to the solid support dueto formation of a stabilized ternary complex. Exemplary flow cells thatcan be used are described, for example, in US Pat. App. Pub. No.2010/0111768 A1, WO 05/065814 or US Pat. App. Pub. No. 2012/0270305 A1,each of which is incorporated herein by reference.

The present disclosure provides systems for detecting nucleic acids, forexample, using methods set forth herein. For example, a system can beconfigured for reactions involving the examination of the interactionbetween a polymerase and a primed template nucleic acid in the presenceof nucleotides to identify one or more bases in a template nucleic acidsequence. Optionally, a system includes components and reagents forperforming one or more steps set forth herein including, but not limitedto, forming at least one stabilized ternary complex between a primedtemplate nucleic acid, polymerase and next correct nucleotide;contacting a ternary complex with stabilizing fluid; detecting thestabilized ternary complex(es); extending the primer of each primedtemplate nucleic acid; and/or identifying a nucleotide, sequence ofnucleotides, or series of base multiplets present in the template.

A system of the present disclosure can include a vessel or solid supportfor carrying out a nucleic acid detection method. For example, thesystem can include an array, flow cell, multi-well plate or otherconvenient apparatus. The vessel or solid support can be removable,thereby allowing it to be placed into or removed from the system. Assuch, a system can be configured to sequentially process a plurality ofvessels or solid supports. The system can include a fluidic systemhaving reservoirs for containing one or more of the reagents set forthherein (e.g. polymerase, primer, template nucleic acid, nucleotide(s)for ternary complex formation, nucleotides for primer extension,deblocking reagents, stabilizing fluid or mixtures of such components).The fluidic system can be configured to deliver reagents to a vessel orsolid support, for example, via channels or droplet transfer apparatus(e.g. electrowetting apparatus). Any of a variety of detection apparatuscan be configured to detect the vessel or solid support where reagentsinteract. Examples include luminescence detectors, surface plasmonresonance detectors and others known in the art. Exemplary systemshaving fluidic and detection components that can be readily modified foruse in a system herein include, but are not limited to, those set forthin US Pat. App. Ser. No. 62/481,289 or Ser. No. 15/922,661, published asUS Pat. App. Pub. No. 2018/0280975 A1; U.S. Pat. Nos. 8,241,573;7,329,860 or 8,039,817; or US Pat. App. Pub. Nos. 2009/0272914 A1 or2012/0270305 A1, each of which is incorporated herein by reference.

Optionally, a system of the present disclosure further includes acomputer processing unit (CPU) that is configured to operate systemcomponents. The same or different CPU can interact with the system toacquire, store and process signals (e.g. signals detected in a methodset forth herein). In particular embodiments, a CPU can be used todetermine, from the signals, the identity of the nucleotide that ispresent at a particular location in a template nucleic acid. In somecases, the CPU will identify a sequence of nucleotides for the templatefrom the signals that are detected.

A useful CPU can include one or more of a personal computer system,server computer system, thin client, thick client, hand-held or laptopdevice, multiprocessor system, microprocessor-based system, set top box,programmable consumer electronic, network PC, minicomputer system,mainframe computer system, smart phone, and distributed cloud computingenvironments that include any of the above systems or devices, and thelike. The CPU can include one or more processors or processing units, amemory architecture that may include RAM and non-volatile memory. Thememory architecture may further include removable/non-removable,volatile/non-volatile computer system storage media. Further, the memoryarchitecture may include one or more readers for reading from andwriting to a non-removable, non-volatile magnetic media, such as a harddrive, a magnetic disk drive for reading from and writing to aremovable, non-volatile magnetic disk, and/or an optical disk drive forreading from or writing to a removable, non-volatile optical disk suchas a CD-ROM or DVD-ROM. The CPU may also include a variety of computersystem readable media. Such media may be any available media that isaccessible by a cloud computing environment, such as volatile andnon-volatile media, and removable and non-removable media.

The memory architecture may include at least one program product havingat least one program module implemented as executable instructions thatare configured to carry out one or more steps of a method set forthherein. For example, executable instructions may include an operatingsystem, one or more application programs, other program modules, andprogram data. Generally, program modules may include routines, programs,objects, components, logic, data structures, and so on, that performparticular tasks set forth herein.

The components of a CPU may be coupled by an internal bus that may beimplemented as one or more of any of several types of bus structures,including a memory bus or memory controller, a peripheral bus, anaccelerated graphics port, and a processor or local bus using any of avariety of bus architectures. By way of example, and not limitation,such architectures include Industry Standard Architecture (ISA) bus,Micro Channel Architecture (MCA) bus, Enhanced ISA (EISA) bus, VideoElectronics Standards Association (VESA) local bus, and PeripheralComponent Interconnects (PCI) bus.

A CPU can optionally communicate with one or more external devices suchas a keyboard, a pointing device (e.g. a mouse), a display, such as agraphical user interface (GUI), or other device that facilitatesinteraction of a use with the nucleic acid detection system. Similarly,the CPU can communicate with other devices (e.g., via network card,modem, etc.). Such communication can occur via I/O interfaces. Stillyet, a CPU of a system herein may communicate with one or more networkssuch as a local area network (LAN), a general wide area network (WAN),and/or a public network (e.g., the Internet) via a suitable networkadapter.

The present disclosure further provides a composition of matter thatincludes one or more of the components set forth herein, for example, inthe context of methods for stabilizing a ternary complex. In particularembodiments, the composition includes a fluid, the fluid containing aternary complex and Li⁺, wherein the ternary complex includes a primedtemplate nucleic acid, a polymerase, and a nucleotide cognate for thenext correct base for the primed template nucleic acid molecule. Thefluid can further contain one or more of betaine, PEI or an inhibitorymetal ion (e.g. Ca²⁺). Optionally, the ternary complex is immobilized ona solid support, for example, via attachment of the template nucleicacid to the solid support. The material for the solid support andattachment means can be any of a variety of those set forth herein orknown in the art. For example, the solid support can include an array offeatures and each feature can include an immobilized ternary complex.The features can have a size, spacing, density or other characteristicin accordance with the disclosure set forth previously herein. One ormore components of the ternary complex, for example, the polymeraseand/or nucleotide, can contain an exogenous label such as a luminophoreor another label set forth herein. The fluid can further includenon-bound nucleotide cognates for at least 1, 2, 3, or 4 bases suspectedof being in the template nucleic acid. The non-bound nucleotide cognatescan have different exogenous labels that distinguish one nucleotide typefrom another. Alternatively or additionally, the fluid can includenon-bound polymerase that optionally contains an exogenous label. Inparticular embodiments, the primed template nucleic acid includes ablocked primer (e.g. a reversibly terminated primer). The concentrationof ternary complex in the fluid can be greater than the concentration ofnon-bound nucleotide and/or greater than the concentration of non-boundpolymerase in the fluid. The fluid can be an aqueous fluid. The fluidcan lack alcohol, oil or other solvents that do not dissolve nucleotidesor polymerases including, for example, the non-miscible fluids set forthin U.S. patent application Ser. No. 16/164,417, which claims priority toU.S. Pat. App. Ser. No. 62/574,308, each of which is incorporated hereinby reference.

This disclosure further provides kits that can be used, for example, forcharacterizing nucleic acids. A kit can include reagents for carryingout one or more of the methods set forth herein. For example, a kit caninclude reagents for producing a stabilized ternary complex when mixedwith one or more primed template nucleic acid. More specifically, a kitcan include one or more of Li⁺, PEI, betaine or an inhibitory metal ion(e.g. Ca²⁺) used in a method set forth herein, including for example,the methods set forth in the Examples section below. In addition, thekit can include at least one nucleotide type and a polymerase that iscapable of forming a stabilized ternary complex. A polymerase that isused for an extension step can also be included in a kit. Thenucleotides, polymerase or both can include an exogenous label, forexample, as set forth herein in the context of various methods.

Accordingly, any of the components or articles used in performing themethods set forth herein can be usefully packaged into a kit. Forexample, the kits can be packed to include some, many or all of thecomponents or articles used in performing the methods set forth herein.Exemplary components include, for example, labeled nucleotides (e.g.extendible labeled nucleotides); polymerases (labeled or unlabeled);nucleotides having terminator moieties (e.g. unlabeled, reversiblyterminated nucleotides); stabilizing fluids such as those containingLi⁺, PEI, betaine and/or an inhibitory metal cation (e.g. Ca²⁺);deblocking reagents and the like as set forth herein and in referencescited herein. Any of such reagents can include, for example, some, manyor all of the fluids, components and/or articles used for performing oneor more of the subsequent steps for analysis of a primed templatenucleic acid. A kit need not include a primer or template nucleic acid.Rather, a user of the kit can provide a primed template nucleic acidwhich is to be combined with components of the kit. Similarly, a kit canexclude one or more of the components set forth herein and, optionally,such excluded components can be provided by an end user.

One or more ancillary reagents also can be included in a kit. Suchancillary reagents can include any of the reagents exemplified aboveand/or other types of reagents useful in performing the methods setforth herein. Instructions can further be included in a kit. Theinstructions can include, for example, procedures for making anycomponents used in the methods set forth herein, performing one or moresteps of any embodiment of the methods set forth herein and/orinstructions for performing any of the subsequent analysis stepsemploying a primed template nucleic acid.

In particular embodiments, a kit includes a cartridge having reservoirsto contain the reagents and further having fluidic components fortransferring reagents from the reservoirs to a detection instrument. Forexample, the fluidic components can be configured to transfer reagentsto a flow cell where stabilized ternary complexes are detected. Anexemplary fluidic cartridge that can be included in a kit (or system) ofthe present disclosure is described in U.S. patent application Ser. No.15/922,661, published as US Pat. App. Pub. No. 2018/0280975 A1, claimingpriority to 62/481,289, each of which is incorporated herein byreference.

Example I Stabilizing Ternary Complexes with an Aqueous StabilizingFluid that Solubilizes Polymerase and Nucleotide

This example demonstrates examination of a blocked primed templatenucleic acid in a manner that assessed binding of each of four differentlabeled nucleotides individually. An aqueous solution containing anagent that was capable of stabilizing ternary complexes was used to: (1)deliver examination solutions containing polymerase, labeled nucleotide,and magnesium cations; (2) separate immobilized ternary complexes fromcontact with non-bound polymerase and labeled nucleotide; and (3)facilitate ternary complex detection over an extended period during animaging wash step. Different nucleotides used in the procedure werelabeled with the same type of fluorescent detectable label and each typeof nucleotide was separately delivered and examined. Alternatively, thenucleotides could have had different detectable labels, respectively,and several different labeled nucleotide types could have been contactedwith the blocked primed template nucleic acid at the same time.

Flow cells containing primed template nucleic acids were prepared asfollows. Template nucleic acid strands synthesized in 12 PCR reactionsusing 5′-biotinylated primers were prepared, and then independentlybound to streptavidin-coated magnetic beads. This resulted in apopulation of 12 bead types, where each bead harbored a homogenouscollection of template strands. Beads used in the procedure had beenfunctionalized with 1 mM NHS-PEG4-TCO in phosphate buffered saline(PBS). Beads harboring immobilized template strands were next flowedover an aminosilane flow cell surface that had been functionalized withtetrazine. The mixture was incubated for one hour to permit covalentattachment of the decorated beads to the functionalized surface withinthe flow cell.

Next, sequencing primers were flowed into the flow cell and allowed tohybridize to the immobilized template strands. Among the aggregatedcollection of beads, there were primed template nucleic acids havingeach of the four bases (i.e., A, C, G or T) as the next templatenucleotide. Reversible terminator nucleotides were then incorporated atthe 3′-ends of the hybridized sequencing primers to create a collectionof blocked primed template nucleic acid molecules. This was accomplishedusing a pH-buffered incorporation mixture that included 10 U/mlTherminator™ polymerase, and 200 nM of unlabeled reversibly terminatednucleotide analogs of dATP, dGTP, dCTP, and dTTP. The reversibleterminator nucleotide used in this illustrative procedure included a3′-ONH₂ reversible terminator moiety that was removable by contact withan acetate-buffered solution containing NaNO₂. A description of thisreversible terminator nucleotide can be found in U.S. Pat. No.7,544,794, the disclosure of which is incorporated herein by reference.

Multiple examination conditions were next screened using the immobilizedbeads attached to the blocked primed template nucleic acid molecules.Reversible terminator moieties on the 3′ nucleotides of the primerstrands precluded nucleotide incorporation during the ternary complexformation and detection steps. This allowed the same base position to beexamined multiple times to determine which conditions supported specificformation and detection of ternary complex. Four different solutionsthat included a polymerase and a single nucleotide labeled on its basewith a Cy5 moiety (i.e., Cy5-dATP, Cy5-dGTP, Cy5-dCTP, or Cy5-dTTP asdescribed in U.S. patent application Ser. No. 15/873,343, which isincorporated herein by reference) were introduced to the flow cellindividually to permit formation of ternary complexes.

After each nucleotide- and polymerase-containing solution was deliveredto permit ternary complex formation, the flow cell was washed withimaging solution to remove non-complexed nucleotide and polymerase.Ternary complexes were detected during the imaging wash step. Thesolution (EXAM solution) providing polymerase and labeled nucleotide forformation of ternary complexes on the immobilized blocked primedtemplate nucleic acid, and the imaging solution (IMG solution) bothincluded 20 mM Tricine buffer (pH 8.42), 50 mM KCl, 0.1% Tween-80, 10 mM(NH₄)₂SO₄, and 3% sucrose. The solution used for forming ternarycomplexes further included 20 U/ml Therminator™ DNA polymerase (NewEngland Biolabs; Ipswich, Mass.), 1 mM MgCl₂, and Cy5-dNTP (400 nM foreach of Cy5-dATP, Cy5-dGTP, and Cy5-dCTP; 800 nM for Cy5-dTTP). Thepolymerase, MgCl₂, and nucleotides were omitted from the imaging fluidstep. Each of the two solutions further included LiCl and/or betaine asstabilizing additives, where the solutions used in the same procedurecontained like concentrations.

After imaging ternary complexes during the imaging wash step, polymeraseand labeled nucleotide were stripped from the ternary complexes bywashing the flow cell with a buffered EDTA-containing guanidiniumthiocyanate solution. The flow cell was then prepared for the next baseexam by flushing with pre-incorporation buffer (20 mM Tricine (pH 8.42),50 mM KCl, 0.1% Tween-80, and 0.1% hydroxylamine). The EXAM and IMGsolutions contained the same core ingredients, except that the imagingfluid did not include polymerase, MgCl₂ or labeled nucleotide. Variableconditions investigated in the procedure involved use of a lithium salt(i.e., LiCl) at either 5 mM or 50 mM, with or without 1 M betaine(N,N,N-trimethylglycine). Concentrations of LiCl and betaine used in thetrials are summarized in Table 1. Results from these procedures arepresented in FIGS. 1A and 1B.

TABLE 1 Variable additions to EXAM Trial and IMG 1 0 mM LiCl; 0M Betaine2 5 mM LiCl; 0M Betaine 3 50 mM LiCl; 0M Betaine 4 0 mM LiCl; 1M Betaine5 5 mM LiCl; 1M Betaine 6 50 mM LiCl; 1M Betaine

FIG. 1A illustrates how lithium cations (e.g., resulting fromdissolution of a lithium salt in water) and/or betaine advantageouslyincreased fluorescent ‘on’ signals without raising fluorescent ‘off’signals for each of the four tested nucleotides. The ‘on’ intensity isthe median pixel count from an imaging camera of all correctly callednucleotides, and ‘off’ intensity is the median pixel count from theimaging camera for the remaining three nucleotides (i.e., the incorrectnucleotides) detected at the same features. Notably, the labeled dTTPnucleotide showed the greatest sensitivity to the different conditions,with the lowest ‘on’ signal being associated with complete absence oflithium cations and betaine. This trend was similar for all fournucleotides. Notably, while either of the tested concentrations of LiClgave substantially saturating ‘on’ intensities, those results werefurther improved by the addition of betaine. This additive effectstrongly suggested that lithium cations and betaine acted to improve thebinding system via different mechanisms.

FIG. 1B graphically confirmed that higher confidence in correct basecalling resulted from the use of lithium and betaine additives. Herepurity values were calculated by dividing the brightest nucleotideintensity value by the sum of the brightest nucleotide intensity and thesecond brightest nucleotide intensity values. To display on a scale of 0to 1, there is an adjustment to subtract 0.5 from the result, and thenmultiply by 2. As indicated, including lithium ions in the imagingsolution used for detecting ternary complexes improved purity values,especially for the labeled dTTP nucleotide. In the absence of lithium,addition of betaine advantageously increased purity values fordTTP-ternary complexes.

Separate experiments (data not shown) demonstrated that increasing theLiCl concentration up to 100 mM, 175 mM or 250 mM also provided high‘on’ signals indicating a stabilizing effect on ternary complexes.However, LiCl at 500 mM resulted in substantial reduction in ‘on’signals and reduced purity, consistent with a destabilization of ternarycomplexes compared to the lower concentrations of LiCl.

These results demonstrate that lithium cations and betaine can provideternary complex stabilization and can be included in an aqueousexamination fluid to permit improved signal to noise ratio whendetecting ternary complex in the presence of non-complexed labelednucleotides.

Example II Stabilization of Ternary Complexes Over Time

This example describes a time titration that demonstrated improveddetection of ternary complexes through washing with imaging buffercontaining lithium and/or betaine for up to 5 minutes.

SBB™ reactions were conducted on blocked 3′-ONH₂ primed template DNAfrom 12 PCR reactions as set forth in Example I except that theextension step was omitted such that the same template position wasrepeatedly examined. Ternary complexes were allowed to form by flushingin EXAM solution as set forth in Example I. IMG solution was thenintroduced to the flow cell and flowed for either 20 seconds, 2½minutes, or 5 minutes at a flow rate of 1 μL/s to wash off non-boundpolymerase-nucleotide complexes prior to detection. The variable washtime and contents of IMG solution that was used for 15 examinationcycles are shown in Table 2.

TABLE 2 Cycle Time LiC1 Betaine 1 20 s 50 mM 1M 2 2.5 m 50 mM 1M 3 5 m50 mM 1M 4 20 s 0 0 5 2.5 m 0 0 6 5 m 0 0 7 20 s 50 mM 0 8 2.5 m 50 mM 09 5 m 50 mM 0 10 20 s 0 1M 11 2.5 m 0 1M 12 5 m 0 1M 13 20 s 50 mM 1M 142.5 m 50 mM 1M 15 20 s 50 mM 1M

As shown in FIG. 2, lithium (50 mM) and betaine (1M) provided increased‘on’ signal intensity for the ternary complexes, shown in cycles 1-3 and13-15. In the absence of lithium and betaine, ternary complexesdissociated and the ability to accurately discriminate correct bases waseliminated (shown in cycles 4-6). The addition of 50 mM lithium (cycles7-9) permitted sequencing, but further addition of 1M betaine incombination with 50 mM lithium (cycles 1-3 and 13-15) showed asynergistic effect, greatly enhanced overall quality.

The results of FIG. 2 demonstrate that betaine helped for a shortduration in stabilizing ternary complex, but after 2½ minutes allternary complexes dissociated (cycle 11). There was a linear decay notedwith overall intensities over the time points of 20 seconds, 2½ minutesand 5 minutes, but as demonstrated by the results shown in FIG. 3 purityvalues remained high and ‘on’ intensities were satisfactory forsequencing. The data collected showed that 50 mM LiCl and 1 M Betaineprovided ternary complex stabilization over time.

Sequencing reactions were conducted with 0 mM, 5 mM and 50 mM LiCl andtheir qualities were observed over the course of 20 cycles. Betaine waspresent in all conditions at a concentration of 1 M. The results shownin FIGS. 4A through 4C demonstrate that increasing concentration ofLiCl, up to 50 mM, increased separation between “on” and “off” signalsand produced more uniform intensities over 20 cycles. The ‘on’intensities were higher with 50 mM LiCl than 5 mM LiCl suggesting thatthe additional lithium aided in forming and stabilizing a greaterpopulation of ternary complexes. FIGS. 5A through 5C show that puritieswere higher with increased concentration of LiCl. Signal to noise ratio(SNR) also improved with increasing LiCl concentration up to 50 mM asdemonstrated by the results shown in FIGS. 6A through 6C. Average errorrates were also lower as the concentration of LiCl increased to 50 mM,as shown in FIGS. 7A through 7C.

Example III Stabilizing Ternary Complexes with an Aqueous StabilizingFluid that Contains Lithium, Calcium or Betaine

This example demonstrates the effects of various combinations oflithium, Calcium and betaine on the stability of ternary complexesduring SBB™ examinations steps.

SBB™ reactions were conducted in flow cells on blocked 3′-ONH₂ primedtemplate DNA from 12 PCR reactions as set forth in Example I except thatthe extension step was omitted such that the same template position wasrepeatedly examined and the following modifications were made. The EXAMsolution contained 20 mM Tricene pH 7.0, 3% sucrose, 50 mM KCl, 10 mMNH₄(SO)₂, 0.1% hydroxylamine, 0.1 mM EDTA, 0.1% Tween-80, 1 mM MgCl₂, 20U/ml Therminator™ DNA polymerase, and Cy5-dNTP (400 nM for each ofCy5-dATP, Cy5-dGTP, and Cy5-dCTP; 800 nM for Cy5-dTTP). The IMG solutioncontained 20 mM Tricene pH 7.0, 3% sucrose, 50 mM KCl, 10 mM NH₄(SO)₂,0.1% hydroxylamine, 0.1 mM EDTA, 0.1% Tween-80. Each of the solutionsfurther contained variable additives as indicated in the tables below.

Ternary complexes were formed by flowing the EXAM solution into flowcells containing the DNA beads. Flow cells were then flushed with 16 μlof IMG solution for 16 seconds. Following the IMG flush, a first imageof the DNA beads in the flow cell was acquired. After a delay of 60seconds, a second image of the beads was acquired from the flow cell.The intensity of ‘on’ and ‘off’ signals were compared between the twoimages to determine ternary complex stability.

Table 3 shows variable contents for 8 examinations that optionallyincluded lithium, calcium or betaine.

TABLE 3 Cycle Variable additives to EXAM and IMG 1 1M Betaine; 50 mMLiCl; 0 mM CaC1₂ 2 0M Betaine; 0 mM LiCl; 0 mM CaC1₂ 3 0M Betaine; 50 mMLiCl; 0 mM CaC1₂ 4 1M Betaine; 0 mM LiCl; 0 mM CaC1₂ 5 0M Betaine; 0 mMLiCl; 0.5 mM CaC1₂ 6 1M Betaine; 0 mM LiC1;0.5 mM CaC1₂ 7 0M Betaine; 50mM LiCl; 0.5 mM CaC1₂ 8 1M Betaine; 50 mM LiCl; 0.5 mM CaC1₂

The percent loss in signal intensity (from the first image to the secondimage) for each cycle is shown in FIG. 8. The percent loss for signalsfrom ternary complexes formed in the presence of the four differentcognate nucleotide types are indicated with individual bars (open barsfor dATP, dotted bars for dGTP, solid bars for dCTP and hatched bars fordTTP). The most drastic signal loss (i.e. lowest stability) was seen forternary complexes incubated in the absence of betaine, Calcium andLithium (see cycle 2). The addition of 1 M betaine had a moderate effecton stabilizing ternary complexes formed with dATP, dGTP and dCTP, butdid not appear to stabilize ternary complexes formed with dTTP (seecycle 4). The addition of 50 mM LiCl alone (see cycle 3) improvedstability and further increase in stability was observed for ternarycomplexes formed with dATP, dGTP and dCTP when both 1M betaine and 50 mMLiCl were present (see cycle 1). The combination of betaine and lithiumalso had a stabilizing effect on ternary complexes formed with dTTP,albeit less pronounced than for the other three nucleotide types.

Continuing with the results in FIG. 8, the addition of 0.5 mM CaCl₂alone (see cycle 5) resulted in an even greater increase in stabilityfor ternary complexes formed with dATP, dGTP and dCTP, compared to theresults with Lithium alone. The presence of 1 M betaine and 0.5 mM CaCl₂(see cycle 6) also stabilized ternary complexes, the stabilization beingslightly better than observed for Calcium alone. The combination of 50mM LiCl and 0.5 mM CaCl₂ (see cycle 7) had a stabilizing effect onternary complexes, especially complexes formed with dATP, dGTP and dCTP.The presence of all three additives, 1M betaine, 50 mM LiCl and 0.5 mMCaCl₂ (see cycle 8) also had a stabilizing effect on ternary complexes,especially complexes formed with dATP, dGTP and dCTP.

FIG. 9 shows purity values obtained from the second images (i.e. after60 second incubation in IMG solution). Cycle 2, run in the absence oflithium, betaine or calcium had the lowest purity scores, indicatingdissociation of the ternary complexes. The addition of betaine aloneresulted in low purity values for ternary complexes formed with dTTP.Cycles carried out in the presence of lithium (with or without betaine)or calcium (with or without betaine) produced complexes having highpurity.

These results demonstrated that the presence of betaine, lithium orcalcium during ternary complex formation and examination improveddetection results, most likely by stabilizing ternary complexes.Independently, lithium and calcium appeared to be most effective.Addition of betaine to lithium or calcium had a synergistic effect inimproving results. Ternary complexes formed with dATP, dGTP and dCTPwere stabilized more significantly under most conditions tested comparedto complexes formed with dTTP. Nevertheless, ternary complexes formedwith dTTP appeared to be stabilized by betaine or lithium, alone or incombinations with betaine.

Several other metals were evaluated for their potential to stabilizeternary complexes in EXAM and IMG solutions. Doubling the concentrationof KCl to 100 mM potassium or adding 50 mM sodium can be used whendetecting ternary complexes, but ternary complexes, especially thoseformed with Cy5-dTTP were less stable than in the presence of similarconcentrations of lithium or calcium. Strontium (10 mM SrCl₂) providedsome stability to ternary complexes formed with purine nucleotides(Cy5-dATP and Cy5-dGTP) but provided little to no stability to ternarycomplexes formed with pyrimidine nucleotides in the conditions tested.Nickel (2.5 mM NiSO₄) and Ytterbium (YbCl₃) did not provide veryeffective stabilization to ternary complexes in the conditions tested.

Example IV Stabilizing Ternary Complexes with Polyethylenimine

SBB™ reactions were conducted on blocked 3′-ONH₂ primed template DNAfrom 12 PCR reactions as set forth in Example I, and with the followingmodifications. The IMG solution included 20 mM Tricine buffer (pH 8.42),50 mM KCl, 0.1% Tween-80, 10 mM (NH₄)₂SO₄, and 3% sucrose along withvariable added reagents as set forth below. The EXAM solution includedthe same fixed and variable reagents as the IMG solution and furtherincluded 20 U/ml Therminator™ DNA polymerase (New England Biolabs;Ipswich, Mass.), 1 mM MgCl₂, and Cy5-dNTP (400 nM for each of Cy5-dATP,Cy5-dGTP, and Cy5-dCTP; 800 nM for Cy5-dTTP).

TABLE 4 Condition Variable additives to EXAM and IMG SOP No variableadditives  0.001 0.001% branched PEI 25k  0.01 0.01% branched PEI 25k0.1 0.1% branched PEI 25k 1.0 1.0% branched PEI 25k

SBB™ reactions were run under the conditions that include variableadditives to IMG and EXAM as specified in Table 4. In all conditions,the beads produced signals indicative of the formation of stabilizedternary complexes. However, it was observed that as the concentration ofPEI increased, the ability to remove PEI and ternary complex from thebeads via the standard wash protocol was decreased. High salt washes (4MNaCl) were effective at removing most of the PEI and base discriminationwas possible after removal of PEI by high salt wash. However, higherbackground was observed after removal of PEI by high salt wash comparedto the background for the SOP condition (i.e. no PEI, standard wash).Heparin was also found to be an effective agent for washing away PEI.Following the use of EXAM and IMG solutions having 0.001% 2 k branchedPEI, the PEI could be removed using wash solution that contained atleast 30 μg/ml heparin (wash solutions having up to 100 μg/ml heparinwere tested and found to be effective).

FIG. 10 shows the results of a stability test carried out as follows.Examination and imaging steps were carried out for beads having aprimed-template with T as the next template nucleotide. For one lane ofthe flow cell, the Exam and IMG buffers included 0.001% branched PEI 25k (labeled as “PEI” in FIG. 10). A control lane was processed inparallel but lacked the added PEI (labeled as “SOP” in FIG. 10). Theimaging step was modified such that a standard image was obtained(labeled “Exam” in FIG. 10), the beads were incubated in the dark for 1minute and a second image was obtained (“1 min” in FIG. 10), and thenthe beads were incubated for another 1 minute before taking a thirdimage (“2 min” in FIG. 10). As is evident from the results of FIG. 10,the presence of PEI provided a significant improvement in stability ofthe ternary complex across the three imaging timepoints.

FIG. 11 shows the results of stability tests carried out as follows.Examination and imaging steps were carried out for beads having avariety of primed-templates. The beads were distributed into 6 differentlanes of a flow cell. The 6 flow cell lanes were processed in parallelbut the EXAM and IMG solutions used for each lane differed as follows:no added PEI (SOP), 0.01% branched PEI 800, 0.025% branched PEI 800,0.05% branched PEI 800, 0.075% branched PEI 800, 0.1% branched PEI 800.The imaging step was modified such that a standard image was obtained,the beads were incubated in the dark for 1 minute and a second image wasobtained (“T=1 min” in FIG. 11), and then the beads were incubated foranother 1 minute before taking a third image (“T=2 min” in FIG. 11). Thedata obtained from several different bead types was averaged such thatall beads that produced a signal indicative of dATP as next correctnucleotide were averaged for the bars labeled A; all beads that produceda signal indicative of dGTP as next correct nucleotide were averaged forthe bars labeled G; all beads that produced a signal indicative of dCTPas next correct nucleotide were averaged for the bars labeled C; and allbeads that produced a signal indicative of dTTP as next correctnucleotide were averaged for the bars labeled T in FIG. 11. As isevident from the results, branched PEI 800 provided improved stabilityespecially as the concentration approached or exceeded 0.075%.

Washes that included 40 μg/ml to 100 μg/ml heparin were effective atremoving branched PEI 800 and ternary complexes from DNA beads(following delivery of EXAM and IMG having 0.1% branched PEI 800).Washes that included 100 μM to 1 mM sodium hexametaphosphate wereeffective at removing branched PEI 800 and ternary complexes from DNAbeads (following delivery of EXAM and IMG having 0.075% branched PEI800).

The results of this Example demonstrated that polyethyleneimine canstabilize ternary complexes. Larger PEI molecules were effective atlower concentrations compared to smaller PEI molecules. However, largerPEI molecules are more difficult to remove from DNA beads than smallerPEI molecules. Smaller PEI molecules can be stripped from DNA beads withheparin and hexametaphosphate.

Throughout this application various publications, patents and/or patentapplications have been referenced. The disclosures of these documents intheir entireties are hereby incorporated by reference in thisapplication.

A number of embodiments have been described. Nevertheless, it will beunderstood that various modifications may be made. Accordingly, otherembodiments are within the scope of the following claims.

What is claimed is:
 1. A method of detecting a primed template nucleicacid, comprising the steps of: (a) providing a mixture comprising aternary complex, the ternary complex comprising a primed templatenucleic acid, a polymerase, and a nucleotide cognate for the next baseof the primed template nucleic acid, wherein the mixture furthercomprises excess nucleotide of the same type present in the ternarycomplex; (b) replacing the excess nucleotide with a fluid comprisingLi⁺; and (c) detecting the ternary complex while it is in contact withthe fluid comprising Li⁺.
 2. The method of claim 1, further comprising(d) identifying the next correct base for the primed template nucleicacid molecule from the result of step (c).
 3. The method of claim 2,further comprising (e) extending the primer of primed template nucleicacid.
 4. The method of claim 3, wherein the primer is extended byaddition of a nucleotide comprising a reversible terminator moiety. 5.The method of claim 1, wherein the primer of the primed templatecomprises a reversible terminator moiety.
 6. The method of claim 5,further comprising modifying or removing the reversible terminatormoiety to deblock the primer.
 7. The method of claim 6, furthercomprising extending the deblocked primer.
 8. The method of claim 7,wherein the deblocked primer is extended by addition of a nucleotidecomprising a reversible terminator moiety.
 9. The method of claim 1,wherein the ternary complex is immobilized on a solid support.
 10. Themethod of claim 1, wherein the nucleotide cognate comprises an exogenouslabel that is detected in step (c).
 11. The method of claim 10, whereinthe mixture comprises nucleotide cognates for at least two differentbase types suspected of being present in the template nucleic acid. 12.The method of claim 11, wherein the nucleotide cognates for thedifferent base types comprise different exogenous labels, respectively.13. The method of claim 1, wherein the polymerase comprises an exogenouslabel that is detected in step (c).
 14. The method of claim 1, whereinthe mixture further comprises excess polymerase of the same type presentin the ternary complex.
 15. The method of claim 14, wherein step (c)comprises replacing the excess polymerase and the excess nucleotide withthe fluid comprising Li⁺.
 16. The method of claim 14, wherein the excesspolymerase is soluble in the fluid.
 17. The method of claim 1, whereinthe excess nucleotide is soluble in the fluid.
 18. The method of claim1, wherein step (b) comprises delivering to the ternary complex a fluidthat comprises Li⁺ and lacks nucleotides of the type present in theternary complex, thereby replacing the excess nucleotide with the fluid.19. The method of claim 1, wherein the Li⁺ is present at a concentrationbetween 1 mM and 100 mM during step (c).
 20. The method of claim 1,wherein the fluid further comprises betaine.
 21. The method of claim 1,wherein the fluid further comprises polyethylenimine.
 22. The method ofclaim 1, wherein the concentration of ternary complex is greater thanthe concentration of free nucleotide in the fluid comprising Li⁺ duringthe detecting in step (c), the free nucleotide being the type ofnucleotide that is present in the ternary complex.
 23. The method ofclaim 1, wherein the concentration of ternary complex is greater thanthe concentration of free polymerase in the fluid comprising Li⁺ duringthe detecting in step (c), the free polymerase being the type ofpolymerase that is present in the ternary complex.